Methods of increasing solubility of poorly soluble compounds and methods of making and using formulations of such compound

ABSTRACT

The subject invention relates to novel soluble forms of planar ring structured organic compounds including flavonoids, and their production. The invention also includes the use of these novel formulations of planar ring structured organic compounds in the preparation of formulations and products. The invention also relates to a wide variety of applications of the formulations of the invention. The subject invention includes novel soluble forms and various formulations of flavonoids. Further, the invention includes novel methods of manufacturing the flavonoid formulations. The invention also relates to a wide variety of applications of the flavonoid formulations.

This application is a continuation of Ser. No. 15/637,370 filed Jun. 29,2017, which is a continuation of Ser. No. 14/107,572 filed Dec. 16, 2013(now U.S. Pat. No. 9,730,953), which is a divisional of Ser. No.13/064,882 filed Apr. 22, 2011 (now U.S. Pat. No. 8,637,569), which is acontinuation-in-part of PCT/US2010/002821 filed Oct. 22, 2010 whichclaims priority to Provisional Application No. 61/253,857 filed Oct. 22,2009, the entire contents of each which are hereby incorporated byreference.

The subject invention relates to novel soluble forms of planar ringstructured organic compounds including flavonoids, and their production.The invention also includes the use of these novel formulations ofplanar ring structured organic compounds in the preparation offormulations and products. The invention also relates to a wide varietyof applications of the formulations of the invention.

BACKGROUND OF THE ART

Flavonoids

The principle plant-derived agents believed to provide protectionagainst cancer are flavonoids and dietary fiber. (Patel, D, et al.,Apigenin and cancer chemoprevention: Progress, potential, and promise,Intl. J. Oncology 2007 January; 30(1): 233-45.) Chemoprevention is afacet of oncology that focuses on the prevention of cancer throughnaturally occurring or synthetic agents.

Flavonoids have been shown to act as free radical scavengers,anti-oxidants, superoxide anions, UV absorbers, and lipid peroxideradicals. Flavonoid compounds are also known to be effective instrengthening collagen structures. Further, flavonoids have been shownto exhibit anti-mutagenic, anti-inflammatory, and antiviral effects.

All flavonoids have the same basic chemical structure, a three-ringedmolecule. Individual flavonoids in a group differ from each other by thenumber and position of substituents (e.g.s the hydroxy, methoxy, orsugar groups). Flavonoids have the planar aromatic ring structuresfollowing general formulas:

Flavonoids comprise approximately 5,000 naturally occurring compounds. Amultitude of other substitutions can be created synthetically, givingrise to the many types of flavonoids.

Many flavonoids are practically insoluble in water and almost allsolvents suitable for pharmaceutical, nutraceutical (fortified foods anddietary supplements), cosmeceutical and medical food applications. Thus,there is a need for methods for enhancing the solubility andbioavailability of these flavonoids including flavones and other planarring structured organic compounds by utilizing acceptable ingredientsand methods.

Solubilization of Active Agents

An active agent is the substance in a composition that is biologicallyactive. Solubility is one of the important parameters to achieve adesired concentration of an active agent in solution for apharmacological response to be elicited. Solubility plays an essentialrole in drug disposition, since it is only that drug in solution that isable to diffuse through a biological matrix or passively transportacross a biological membrane. The maximum rate for drug absorption is aproduct of permeability and solubility. Drug efficacy can be severelylimited by poor aqueous solubility. It is commonly recognized in thepharmaceutical industry that on average more than 40% of newlydiscovered drug candidates are poorly water-soluble. Poor solubilityproperties of drugs leads to ineffective absorption from the site ofadministration; which has been designated as an important part of thehigh clinical failure due to poor pharmacokinetics (Liu, R.,Introduction, In: Liu, R., (Ed.), Water-Insoluble Drug Formulation,2^(nd) ed., 2008, CRS press, New York).

There are many techniques that have been used to enhance the solubilityof active agents. They include the use of complexing ligands such ascyclodextrins which increase the aqueous solubility of poorly solubleactive agents by encapsulating them into the hydrophobic bucket shapedcavity of the cyclodextrin molecule; the nano-sizing of active agents tonano-sized crystals; the use of active agent salt forms (which tend toexhibit improved aqueous solubility and dissolution properties incomparison to the original active agent); and the alteration of theactive agents pH microenvironment; etc.

When formulating topical drug products, the active pharmaceuticalingredient is typically added to solvent or solvent mixture to dissolveor disperse the ingredient with mixing or homogenation. Heat, typicallyin the range of 35° C. to 70° C., has been used to melt low meltingpoint excipients of the formulation to aid in mixing. Heat is kept to aminimum to prevent decomposition, separation upon reaching the cloudpoint, or loss due to evaporation of any of the excipients or activeingredient.

When forming an emulsion, the active agent is added to either theaqueous or lipid phase of the formulation (generally that phase with thehighest solubility for the active agent). One or both phases can beheated in the same range of temperatures noted above either prior to orduring mixing of the two phases to make the emulsion.

Dimethyl sulfoxide (DMSO) has been widely used in both in vitro and invivo studies as a solvent for many water insoluble compounds includingapigenin. However, due to toxicity and “taste” concerns, dimethylsulfoxide is not widely used as a solvent when a topical formulation isconsidered for human applications. Nearly all apigenin animal studiesdevoted to anti-skin cancer topical treatments have utilized dimethylsulfoxide (DMSO) as the solvent of choice due to apigenin's poorsolubility in water (<0.005 milligram per milliliter (mg/ml)) and otheraqueous solvents. (Li et al, Evaluation of Apigenin and [G-³ H],Apigenin and analytical method development, J. of PharmaceuticalSciences. Vol. 86, No. 6, June 1997).

Skin Cancer

The development of skin cancer is a major global public health threat.Ultraviolet (UV), e.g., solar ultraviolet B (UVB) and solar ultraviolet(UVA), radiation are the main causes of skin cancer. The incidences ofbasal cell carcinoma, squamous cell carcinoma, and melanoma continue torise despite the advent and use of sunscreen agents with high SPFconstituents. Early detection and treatment are essential in improvingsurvival rates, yet skin cancer is a cancer that is largely preventablealtogether. Current sunscreen formulations have proven inadequate forfully protecting persons from the DNA-damaging effects of UV radiation.Sunscreen usage may sometimes create a false sense of safety asindividuals may over expose themselves to sunlight.

Studies have demonstrated that flavones possess anti-oxidant,anti-mutagenic, anti-carcinogenic, anti-inflammatory,anti-proliferative, and anti-progression properties. (Patel, D, et al.,Apigenin and cancer chemoprevention: Progress, potential, and promise,Intl. J. Oncology 2007 January; 30(1): 233-45.) In addition, Birt andcoworkers used an in vivo mouse model to demonstrate that topicalapplication of apigenin prior to UVB-irradiation significantly reduced,by up to 90%, the incidence of skin cancer. (Birt et al.,Anti-mutagenesis and anti-promotion by apigenin, robinetin andindole-3-carbinol, Carcinogenesis, June 1986; 7: 959-963) Other groupshave demonstrated apigenin's ability to protect mice against coloncancer. (Wang et al, Cell cycle arrest at G2/M and growth inhibition byapigenin in human cell colon carcinoma cell lines, MolecularCarcinogenesis, 28: 102-110 (2000))

Loss of G1/S and/or G2/M cell cycle checkpoint controls leads totransformation and cancer progression. Initiation and progressionthrough the cell cycle is largely controlled by proto-oncogenes thatpromote cell proliferation and tumor suppressor genes that function toslow or halt cell growth. Mutations in either proto-oncogenes and/ortumor suppressor genes predispose cells to a compromised G1/S checkpointby shortening the length of time spent in G1 or G2/M.

Researchers have found that apigenin induces reversible, cell-cyclearrests at G1 and G2/M phase of the cell cycle. It was furtherdiscovered that apigenin mediates an inhibition on the cell cyclethrough multiple mechanisms including direct and indirect inhibition ofthe mitotic kinase p34cdc2, as well as the induction of the cell cycleinhibitor p21WAF1 in a p53-dependent manner. Theoretically, the neteffect allows UV induced DNA mutations to be repaired properly prior tocell division. (Lepley D M, et al., The chemopreventative flavonoidapigenin induces G2/M arrest in keratinocytes, Carcinogenesis, 17,2367-75 (1996))

Other Skin Disorders

Kang, Ecklund, Liu & Datta, (Arthritis Research & Therapy 2009, Vol. 11)taught that increasing the bioavailability of dietary plant-derivedCOX-2 and NF-κB inhibitors, such as apigenin, could be valuable forsuppressing inflammation in lupus and other Th17-mediated diseases likepsoriasis. Apigenin, a non-mutagenic dietary flavonoid, suppresses lupusby inhibiting autoantigen presentation for expansion of autoreactive Th1and Th17 cells.

Other Diseases

As is typical for phenolic compounds, flavonoids act as potentantioxidants and metal chelators. They also have long been recognized topossess antiinflammatory, antiallergic, hepatoprotective,antithrombotic, antiviral, and anticarcinogenic activities.

The flavones and catechins are very powerful flavonoids for protectingthe body against reactive oxygen species (ROS). Body cells and tissuesare continuously threatened by the damage caused by free radicals andROS which are produced during normal oxygen metabolism or are induced byexogeneous damage. The anti-inflammatory activity of flavonoids in manyanimal models has been reported. Flavones/flavonols such as apigenin,luteolin, kaempferol, quercetin, myricetin, fisetin were reported topossess Lipoxygenase (LO) and Cyclo-oxygenase (COX) inhibitoryactivities. Jachak S M Natural products: Potential source of COXinhibitors. CRIPS 2001; 2(1):12-15.

PCT/US2006/020905 to Doseff discloses methods of treating inflammationwith apigenin or its derivatives.

US Patent application US 2008/0227829 to Hammerstone discloses methodsof treating subjects with a neurogenic compound including apigenin.

U.S. Patent application US 2007/0154540 to Park et al discloses the useof apigenin as a chondroregenerative agent for the treatment ofosteoarthritis.

U.S. Patent application US 2007/0189680 to Bing-Hua et al discloses theuse of apigenin for chemoprevention and chemotherapy combined withtherapeutic reagents.

U.S. Patent application US 2006/0067905 to Lintnera et al discloses theuse of apigenin as a vasodilatory agent for treating baldness.

Research studies have provided evidence that apigenin plays a criticalrole in the amelioration of the pathogenetic process of asthma. Recentepidemiological studies reported that a low incidence of asthma wassignificantly observed in a population with a high intake of flavonoids.

Hyaluronic Acid

Hyaluronic acid (HA) is an anionic, nonsulfated glycosaminoglycandistributed widely throughout connective, epithelial, and neuraltissues. It is unique among glycosaminoglycans in that it isnonsulfated, forms in the plasma membrane instead of the Golgi, and canbe very large, with its molecular weight often reaching the millions.One of the chief components of the extracellular matrix, HA contributessignificantly to cell proliferation and migration.

Polysaccharides such as HA are relatively complex carbohydrates.Polysaccharides are polymers made up of many monosaccharides joinedtogether by glycosidic bonds. The polysaccharides are therefore large,often branched, macromolecules. Polysaccharides have been useful incosmetic and medical applications. For example, HA finds use as astructure stabilizing filler for dermal applications. Apigenin hasantihyaluronidase activity; thereby inhibiting the breakdown ofhyaluronic acid. (Kuppusamy et al., Structure-activity studies offlavonoids as inhibitors of hyaluronoidase, Biochem Pharmacol, 40,397-401 (1990).

U.S. Patent application 2005/0271692 to Gervasio-Nugent et al disclosestopical cosmetic compositions which include flavonoids and hyaluronicacid.

U.S. Patent application 2006/021625 to Morariu discloses topicalformulation and methods of use for improving the appearance of agedskin. Preferred components include flavonoids such as apigenin andhyaluronic acid.

Polysorbate Surfactants

Polysorbates (commercially also known as Tweens) are nonionicsurfactants and emulsifiers derived from polyethoxylated sorbitan andfatty acids. They are often used in foods and in cosmetics to solubilizeessential oils into water-based products. The polysorbates are viscous,water-soluble pale yellow liquids. Polysorbates also help to formemulsions by reducing the surface tension of the substances to beemulsified. Polysorbates have been recognized for their ability to helpingredients to dissolve in a solvent in which they would not normallydissolve. Polysorbates function to disperse oil in water as opposed towater in oil. Polysorbates are produced by reacting the polyol,sorbitol, with ethylene oxide. The polyoxyethylenated sorbitan is thenreacted with fatty acids obtained from vegetable fats and oils such asstearic acid, lauric acid, and oleic acid. Surfactants that are estersof plain (non-PEG-ylated) sorbitan with fatty acids are usually referredto by the name Span.

U.S. Pat. No. 7,329,797 to Gupta discloses antiaging cosmetic deliverysystems which includes the use of flavonoids including apigenin as ananti inflammatory agent and polysorbate surfactants as emulsifyingagents,

U.S. Patent Application 2006/0229262 to Higuchi et al disclosepharmaceutical compositions for the treatment of infections fortreatment of infections with a drug resistant bacteria infections withagents including flavonoids such as apigenin as an active ingredient andpolysorbates as emulsifying agents.

U.S. Pat. No. 6,048,566 to Behnam discloses non-alcoholic beverages andprocesses of making them. The patent discloses mixing ubiquinone Q10 anda polysorbate solubilizer.

Polyethylene Glycols

Poly(ethylene glycol) (PEG), otherwise known as poly(oxyethylene) orpoly(ethylene oxide) (PEO), is a synthetic polyether that is readilyavailable in a range of molecular weights (MW). Materials withMW<100,000 are usually called PEGs, while higher molecular weightpolymers are classified as PEOs. These polymers are amphiphilic andsoluble in water as well as in many organic solvents. Low molecularweight (MW<1,000) PEGs are viscous and colorless liquids, while highermolecular weight PEGs are waxy, white solids with melting pointsproportional to their molecular weights to an upper limit of about 67°C. PEG has been found to be nontoxic and is approved by the FDA for useas a surfactant or as a carrier in different pharmaceuticalformulations, foods, and cosmetics. Most PEGs with MW>1,000 are rapidlyremoved from the body unaltered with clearance rates inverselyproportional to polymer molecular weight. This property, combined withthe availability of PEGs with a wide range of end-functions, contributesto the wide use of PEGs in biomedical research: drug delivery, tissueengineering scaffolds, surface functionalization, and many otherapplications.

In view of the foregoing, it is most desirable to improve the solubilityof poorly soluble compounds including flavonoids. It is also desirableto incorporate flavonoids, such as the flavones apigenin and luteolin,as part of topical formulations to aid in the prevention and/ortreatment of skin damage or skin cancer resulting from the effects ofsun exposure and also to provide a skin treatment composition useful inthe treatment of a variety of dermatological conditions.

SUMMARY OF THE INVENTION

The subject invention relates to a composition comprising i) a planarring structured organic compound, and ii) a heat stable solubilizingcompound, wherein the concentration of the planar ring structuredorganic compound is greater than the saturation concentration of theplanar ring structured organic compound in said heat stable solubilizingcompound, and said composition is not supersaturated. The heat stablesolubilizing compound is typically a nonionic surfactant such as apolysorbate. The composition also typically includes a carrier.

The subject invention relates to compositions comprising a planar ringstructured organic compound, such as a flavonoid, and a heat stablesolubilizing compound such as a surfactant, wherein the composition isformed by: mixing the planar ring structured organic compound and thesolubilizing compound to an elevated temperature (typically >100° C.)where said planar ring structured organic compound is dissolved in thesolubilizing compound. When the dissolved mixture is cooled to ambienttemperatures, the planar ring structured organic compound remainsdissolved even at concentrations exceeding the ambient temperaturesaturation concentration level.

The composition optionally includes an alcohol selected from the groupconsisting of ethanol, small-chain alcohols (such as isopropyl andbenzyl alcohol), ethoxydiglycol (diethylene glycol monoethyl ether orTranscutol), propylene glycol, hexylene glycol, butylene glycol,dipropylene glycol, glycerin, water, saline, DMSO, isopropyl myristate,mineral oil, low viscosity surfactants, and dimethyl isosorbide.

In a preferred embodiment, the composition is a pharmaceuticalcomposition and the carrier is a pharmaceutically acceptable carrier.The composition can include hyaluronic acid, and/or a penetrationenhancer. In one embodiment the composition is in the form of anemulsion or microemulsion. The composition can be, for example, apharmaceutical composition, a nutraceutical (fortified foods or adietary supplement), cosmeceutical, a food supplement, or medical food.

Another embodiment of the invention is a patch for application of aplanar ring structured organic compound, such as a flavonoid,transdermally comprising a substrate having two sides, a first sidehaving a composition of the invention and an adhesive (with a releaseliner), and a second side with a material which is impermeable to thecomposition and adhesive on the first side. In another embodiment, thepatch comprises a substrate having two sides, the first side having aliquid reservoir containing a composition of the invention and asemipermeable membrane and an adhesive layer attached to the undersideof the semipermeable membrane (with a release liner) and a second sidewith a material which is impermeable to the composition and to theadhesive on the first side.

Another embodiment of the invention is a method of preparing asolubilized planar ring structured organic compound, such as a flavonoidcomposition comprising: mixing a planar ring structured organiccompound, such as flavonoid particles, with a heat stable solubilizingcompound, such as a surfactant, to form a mixture, heating the mixtureto a temperature where the planar ring structured organic compound issolubilized, and cooling the solution. In an advantageous embodiment,the heat stable solubilizing compound is a nonionic surfactant.Typically, the mixture is stirred while heating, and up to 10 wt % of aplanar ring structured organic compound, such as a flavonoid compound,is added. In an advantageous embodiment, the surfactant is apolysorbate. After the heating or cooling step is the step of adding thesolution to a dermatological, oral, injectable, periodontal, dermalpatch, or aerosol carrier. A small chain alcohol selected from the groupconsisting of ethyl alcohol, isopropyl alcohol, benzyl alcohol,ethoxydiglycol and dimethyl isosorbide, can be added to the solution.

The invention also relates to method of increasing the solubility of apoorly soluble planar ring structured organic compound compositioncomprising: i) mixing the planar ring structured organic compound with aheat stable solubilizing compound to form a mixture, ii) heating themixture to a temperature where the planar ring structured organiccompound particles are solubilized to form a solution, and iii) coolingthe solution.

The invention also relates to a method of reducing and/or preventing theeffects of sun exposure comprising applying a therapeutically effectiveamount of a sunscreen formulation to the skin comprising a solubilizedplanar ring structured organic compound, such as a flavonoid, and acarrier that permits delivery of the planar ring structured organiccompound, such as a flavonoid, to the stratus corneum and the epidermis.In another embodiment, the formulation additionally comprises mineraloxides to provide additional ultraviolet sun exposure protection.

In another embodiment, the invention relates to a method of treating theeffects of sun exposure comprising applying a therapeutically effectiveamount of a formulation to sun damaged skin comprising a solubilizedplanar ring structured organic compound, such as a flavonoid, and acarrier that permits delivery of the flavonoid to the stratus corneumand the epidermis.

In another embodiment, the invention relates to a method of reducing thelikelihood of or treating cancer in a mammal comprising administering toa mammal in need of such treatment a prophylactic amount or atherapeutically effective amount of a formulation of the invention.

In another embodiment, the invention relates to a method of treatinginflammation in a mammal comprising administering to a mammal in need ofsuch treatment a therapeutically effective amount of a formulation ofthe invention.

In another embodiment, the invention relates to a method of restoringnormal skin barrier function in a mammal comprising administering to amammal in need of such treatment a therapeutically effective amount of aformulation of the invention such as a flavonoid formulation.

In another embodiment, the invention relates to a method of treating askin disease or disorder such as acne, alopecia, dermal sensitizationand irritation, dry skin (xerosis, ichthyosis), fungal infections, androsacea, contact dermatosis, in a mammal comprising administering to amammal in need of such treatment a therapeutically effective amount of aformulation of the invention.

In another embodiment, the invention relates to a method of treatingautoimmune disease such as psoriasis, lupus, and arthritis in a mammalcomprising administering to a mammal in need of such treatment atherapeutically effective amount of a formulation of the invention.

In another embodiment, the invention relates to a method of treatingallergies, asthma, atopic dermatitis/eczema comprising administering toa mammal in need of such treatment a therapeutically effective amount ofa formulation of the invention.

In another embodiment, the invention relates to a method of treating orreducing the likelihood of a TNFα related disease in a mammal comprisingadministering to a mammal in need of such treatment a therapeuticallyeffective amount or a prophylactic amount of a flavonoid formulation ofthe invention.

In another embodiment, the invention relates to a method of treating orreducing the likelihood of an IL-113 related disease in a mammalcomprising administering to a mammal in need of such treatment atherapeutically effective amount or a prophylactic amount of a flavonoidformulation of the invention.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows surfactant classification according to their head:nonionic, anionic, cationic and amphoteric.

FIG. 2 is a mass spectroscopy plot indicating insignificant chemicalcomposition differences between the unprocessed polysorbate 80 controlsample and the thermally treated polysorbate 80 sample.

FIG. 3 is a graphical plot illustrating the apigenin content depositedwithin the epidermal, dermal and receptor fluid segments of humantissues for several applied topical formulations containing variousapigenin concentrations.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention relates to new formulations of planar ringstructured organic compounds, such as a flavonoids. Included arepharmaceutical compositions, nutraceuticals, cosmeceuticals, foodsupplements, and medical foods, containing such formulations, as well asmethods for making and using the same.

Solubility is an important parameter for the topical and oralbioavailability of poorly soluble planar ring structured organiccompound, such as a flavonoid. Dissolution of a drug can be the ratedetermining step of a poorly water soluble drug for oral absorption andtopical absorption. Drug solubility is also the basic requirement forthe formulation and development of different dosage forms where the drugis intended to be in solution. Because of solubility problems associatedwith many planar ring structured organic compounds, such as aflavonoids, their bioavailability is limited and hence solubilityenhancement becomes necessary. The methods of making and using novelsoluble forms of poorly soluble planar ring structured organic compoundstaught in this disclosure, address the enhancement of poorly solublering structured organic compounds.

The subject invention relates to adding a planar ring structured organiccompound, such as a flavonoid, to a heat stable surfactant and thenheating the mixture up to an elevated temperature (typically >100degrees C.), not exceeding the boiling point or decomposition point ofeither the active agent (e.g. flavonoid) or the heat stable solubilizingagent (e.g. surfactant), and then cooling the mixture. It wasunexpectedly observed that this process can enhance dissolution, and asignificantly higher concentration of the planar ring structured organiccompound in solution with the surfactant can be achieved. Furthermore,the resulting solution “concentrate” is not supersaturated. The molarratio of active agent to solubilizing agent is typically 1:2 to 1:5, andat times much greater, e.g. 1:2 to 1:20 depending on the activeagent/surfactant combination. While not wishing to be bound by theory,it is believed that the heat necessary to facilitate dissolution isneeded to overcome the bonds associated with self-associated stacking ofthe planar ring structured organic compounds—which limits theirsolubility. Upon dissolution of these planar ring structured organiccompounds with heat in the heat stable solubilizing agent, thesolubilizing molecules coat or sandwich these planar compounds—ie theheat stable solubilizing agent and the planar compound “associate” orform a “complex.” Upon cooling to room temperature, the concentrates arenot supersaturated solutions even though the concentrations of thecompounds are greater than their saturation concentration at ambientconditions—room temperature (temperature below that necessary toovercome the planar ring structured organic compound self-associatedstacking forces). The concentrate is stable and the compounds (or activeagents) stay in solution for periods of time sufficient for makingformulations from the concentrates.

These planar ring structured organic compound concentrates can be usedto formulate compositions with a higher concentration of the compoundsin solution than that achievable without the use of the concentrates.That is, these concentrates can be used to prepare formulations thathave the planar ring structured organic compounds at a higherconcentration in solution than that achievable by a) use of standardformulation preparation techniques, orb) mixing of the planar ringstructured organic compounds with a surfactant and excipients alltogether, and then heating to an elevated temperature.

I—Compounds of the Invention

The present invention relates to molecules that are planar with at leastone ring structure that allow for a stacking arrangement. The ringstructure can be an aromatic structure. Those molecules that do stackcharacteristically have a high melting point and demonstrate poorsolubility. It is the poor solubility characteristics that typicallylimit practically for use of these compounds in topical products anddrug formulations. As used herein, the term “poor solubility” meanshaving a solubility in water or oil less than 1 mg/ml, and particularlyless than 0.1 mg/ml.

Examples of planar ring structured organic compounds with poorsolubility include the following important classes of active agents:

Class 1. The camptothecin analogs are used as antineoplastic agents andare sparingly soluble in water. The methods of the present invention canbe applied to solubilize these agents. Included are camptothecin,topotecan and irinotecan and compounds with other substitutions for R10,R9 and R7

R10 R9 R7 Camptothecin H H H Topotecan OH (CH₃)₂NHCH₂ ⁻ H Irinotecan

H CH₃CH₂ ⁻Class 2. The guanidine nucleosides used as antivirals and in particularantiherpes treatments. Such agents have limited water solubility.Included are acyclovir, penciclovir, ganciclovir, and compounds withother substitutions for R1.

Class 3. The benzodiazepines are CNS active drugs for depression andanxiety. These tricyclic agents are sparingly soluble in water and canbe solubilized using the methods of the invention. Such compoundsinclude alprazolam, oxazepam, and all related agents of the generalformula.

Class 4. The tertiary anime tricyclic antidepressants can be madesoluble with treatment with mineral acids and as acid salts. The methodsof this invention solubilize these agents without the use of acids. Suchcompounds include amitriptyline, clomipramine, doxepin and all relatedcompounds such as maprotiline and protriptyline

R1 X1 X2 Amitriptyline H CH₂ C═CCH₂CH₂N(CH₃)₂ Clomipramine Cl CH₂NCH₂CH₂CH₂N(CH₃)₂ Doxepin H O C═CCH₂CH₂N(CH₃)₂Class 5. The tricyclic phenothiazines are used as antipsychotics and canbe solubilized by the methods of the invention without the use of strongacids. Such compounds include chlorpromazine, chlorprothixene andrelated tricyclics

X R1 R2 Chlorpromazine N CH₂CH₂CH₂N(CH₃)₂ Cl Chlorprothixene C═CCH₂CH₂N(CH₃)₂ Cl Thioridazine N

SCH₃Class 6. The tricyclic iminostilbenes are used as anticonvulsants andcan be solubilized by the methods of the invention. Such compounds arecarbamazepine, oxcabazepine and all related tricyclic iminostilbenes.

Class 7. The method of the present invention can also be applied tononfused ring systems. Such compounds include dilantin and thedicarbamates, felbamate and levetiracetam which are all very waterinsoluble anti-seizure medications. Other related compounds are alsoincluded.

Class 8. Many of the dihydrofolic acids analogs are sparingly soluble inwater. The method of the present invention can be applied to this classof agents including methotrexate and trimetrxate which are used forpsoriasis and autoimmune diseases. Related compounds are included.

Class 9. The sulfa antibacterials can be solubilized by the methods ofthe present invention. Such compounds include sulfamethoxazole,sulfadoxine and all related sulfa antibacterial compounds.

Class 10. Several sulfa drugs are also carbonic anhydrase inhibitors andare useful for the treatment of glaucoma, ulcers, and as diuretics.These compounds have poor water solubility and can be solubilized by themethods of the present invention. Such compounds include ethoxzolamide,acetazolamide, and the anticonvulsant sultiam.

Class 11. The anthracycline anticancer agents are tetracyclines withpoor water solubility. Such compounds include doxorubicin (adriamycin),daunorubicin, epirubicin, idarubicin, and all related compounds.

Class 12. The epipodophyllotoxins are useful and anticancer agents. Themethods of the present invention can be used to solublize compounds inthis class. Such agents include etoposide, teniposide and all relatedcompounds.

Class 13. Certain of the pyrimidine nucleoside analogs are poorlysoluble in water and are useful as antineoplastic agents, for psoriasis,or to inhibit the growth of DNA viruses.

Compounds in this class are 5-fluorouracil (5-FU), floxuridine,gemcitabine, zidovudine, stavudine, and all related compounds.

R1 R2 R1 R2 Floxuridine

F Gemcitibine

H Idoxuridine

I Zalcitabine

H Trifluridine

CF₃ Lamivudine

HClass 14. Similar to the pyrimidine nucleosides, the adenine andguanidine based chemotherapeutics which are useful vs DNA viruses, arealso sparingly soluble in water unless combined with strong acids. Themethod of the present invention can solublize these agents in waterwithout the use of strong acids. Compounds included in this classinclude didanosine and abacavir and all related compounds.

Class 15. Certain selective estrogen receptor modulators have found useas treatments for breast cancer, osteoporosis and contraception. Many ofthese poly phenyl agents have low solubility in water unless combinedwith strong acids. The methods of the present invention are useful forincreasing the water solubility without the use of acids. Such agentsinclude tamoxifen, clomiphene, lasofoxifene and all related agents.

Class 16. Still other selective estrogen receptor modulators belong toanother structural class. One such agent is raloxifene which is usefulfor treating osteoporosis and other estrogen related diseases. In theabsence of strong acids or very low PH raloxifene is sparingly solublein water. The methods of the present invention can increase thesolubility of this and related agents.

Class 17. The progesterones are a class of steroidal hormones and agentsaffecting the female reproduction cycle. Such agents include megesterolacetate and medroxyprogesterone and all related agents and are used inmany areas of human and animal health. All the compounds in this classare sparingly soluble in water. Solubility in water can be improved byapplication of the methods of the present invention.

Class 18. The estrogens are a class of steroid hormones that affect thefemale reproductive cycle, development, and maturation. Such compoundsinclude estradiol, estradiol valerate, mestranol, estrone and allrelated analogs. All of the compounds in this class are sparinglysoluble in water. Solubility can be improved by application of themethods of the present invention.

R1 R2 R3 R4 Estradiol H H H H Estradiol Valerate H H CO(CH₂)₃CH₃ HEstriol H H H OH Mestranol CH₃ C≡CH H H

Class 19. The testosterones and the nortestosterones are steroidhormones affecting the reproductive cycle in humans and which alsoaffect growth, development and maturation and related processes. Theanabolic steroids also belong to this class. Such agents includemethyltestosterone, oxandrolone, danazol and a many related compoundswithin the class. Like most of the steroids, these compounds aresparingly soluble in water and may be solubilized by the methods of thepresent invention.

Class 20. The corticosteroids are useful for the treatment ofinflammation, carbohydrate and lipid metabolic syndromes and for otherdisease states. Such compounds include hydrocortisone, fludrocortisone,dexamethasone and all related agents. These corticosteroids aresparingly soluble in water but can be made more soluble by applicationof the methods of the present invention.

R1 R2 R3 R4 Hydrocortisone OH Single Bond H H Fludrocortisone OH SingleBond F H Dexamethasone OH Double Bond F CH₃ Cortisone C═O Single Bond HHClass 21. The glitizones are a class of compounds used to treathyperglycemia and insulin resistance. Such compounds includerosiglitazone, pioglitazone, troglitizone and all related analogs. Thesecompounds are only sparingly soluble in water, but the solubility can beimproved by application of the methods of the present invention.

Class 22. The quinone class of agents which include atovaquone,buparvaquone and parvaquone are useful as antiprotozoal drugs. Theseagents and all of their analogs tend to be sparingly soluble in water.They can be solubilized by application of the methods of the presentinvention.

Class 23. The quinoline class of agents which include mefloquine,primaquine, cloroquine and all related analogs are useful asantimalarials. These agents are soluble in strong acid or as their acidsalts. The methods of the present invention can solublize these agentsin water without the use of strong acids.

Class 24. Piroxicam and meloxicam and their related analogs are usefulto treat pain, swelling, and other related symptoms. These agents aresparingly soluble in water. These agents and all related analogs can besolubilized by application of the present invention.

Class 25. The propionates are a class of agents useful for treatingpain, fever, and inflammation. Members of this class include ibuprofen,naproxen, fenoprofen, nabumetone and many related analogs. Thesecompounds are weak acids and are sparingly soluble in water. They may besoluble in strong alkali and high pH. The methods of the presentinvention solublize these agents without the need for strong alkali orhigh pH

Class 26. Multi-ring anti-inflammatory agents such as indomethacin,sulindac and all of their analogs are sparingly soluble in water withoutthe addition of strong alkali or high pH. Using the methods of thepresent invention, these agents can be solubilized without the use ofalkali or high pH.

Class 27. The indole based antiemetics which include ondansetron,dolasetron, granisetron, and all their related analogs are 5-HT3receptor antagonists. These agents tend to be sparingly soluble inwater. They and their related analogs can be solubilized by applicationof the methods of the present invention.

Class 28. The azole antifungals are an important therapeutic class ofagents including clotrimazole, miconazole, sulconazole, ketoconazole andall of their related anaologs. These agents tend to be sparingly solublein water. They can be solubilized by application of the methods of thepresent invention.

Class 29. The benzimidazoles omeprazole, lasoprazole, and of the relatedanalogs have utility as proton pump inhibitors. These agents aresparingly soluble in water but can be solubilized by application of themethods of the present invention.

Class 30. Compounds with various aromatic rings lacking sites forsolubilization with strong acids or strong alkali can be solubilized byapplication of the methods of the present invention. Examples of suchagents that can be solubilized are:

Class 31. Compounds of the quinolone class of antibacterials such asciprofloxacin, moxifloxacin and ofloxacin are sparingly soluble inwater. These compounds have heterocyclic side chains which bear a basicnitrogen, which can form salts with strong acids. The salts increasesolubility but even the salts are sometimes less soluble than desired.The methods of the present invention will increase the water solubilityof these compounds without the use of strong acids. For cases wherethere is no basic nitrogen or only weakly basic nitrogens, the methodsof the present invention will still solublize these agents. One suchexample is WCK771. The generic structure describes some of the compoundswhere the methods would apply.

Class 32. Certain of the compounds which act as local anesthetics suchas benzocaine, mepivacaine, lidocaine and related structures aresparingly soluble in water without the addition of strong acids or lowpH. Application of the methods of the present invention increase thesolubility of such compounds without the use of strong acids or low pH.

Class 33. Capsaicin and its related compounds are useful for treatmentof pain or injury. Capsaicin is sparingly soluble in cold water.Application of the methods of the present invention increases thesolubility of capsaicin and related compounds.

Class 34. The aryl acetic acids such as tolmetin, ketorolac, diclofenacand their related structures are useful as anti-inflammatory agents andfor treatment of pain. They are sparingly soluble in water. At higher pHor with treatment with base the solubility is improved. The methods ofthe present invention increase the solubility of these agents and theiranalogs in water without the use of base or elevated pH.

Class 35. Several anti-inflammatory and pain agents belong to a broadclass of diphenyl heterocycles such as rofecoxib, celecoxib,sulfinpyrazone, phenylbutazone and related compounds. These compoundsare sparingly soluble in water. Application of the methods of thepresent invention increase the solubility of these and related compoundsin water.

Class 36. The statins are a well known class of agents which are usefulto treat hyperlipidemia and related ailments. This class of compoundsincludes lovastatin, atorvastatin, cerivastatin and all relatedstructures. These compounds are sparingly soluble in water and evenseveral of the acid salts are not freely soluble. Application of themethods of the present invention increases the solubility in water forthese and the compounds in this class.

Class 37. Fibrates such as fenofibrate are poorly soluble compounds.Application of the methods of the present invention increase thesolubility of these compounds in water.

Below are chemical structures and chemical/physical properties of someother poorly soluble planar ring structured organic compounds:

TABLE I PROPERTIES OF SOME PLANAR ACTIVE AGENT Active Agents Usage CAS #MW MP-° C. MEFENAMIC Pain Relief/NSAID 61-68-7 241      230 ACIDDICLOFENAC Pain Relief/NSAID 15307-86-5 318      283 SODIUM⁽¹⁾DICLOFENAC Pain Relief/NSAID 15307-79-6 296 ~⁽²⁾177 ACID Note:⁽¹⁾Synthesized from Sodium Diclofenac-See Diclofenac Section for details⁽²⁾J Pharmaceut Sci, 6(3): 352-359, 2003Polyphenols Including Flavonoids

The chemical structures of some commonly occurring plant planar ringstructured flavonoids are listed below.

TABLE II CHEMICAL STRUCTURES OF SOME COMMONLY OCCURING PLANT FLAVONOIDSRepresentative Structure flavonoids

R1 = H, R2 = OH: Apigenin R1 = R2 = OH: Luteolin

R2 = OH, R1 = R3 = H: Kaempferol R1 = R2 = OH, R3 = H: Quercetin R1 = R2= R3 = OH: Myricetin

R1 = H: Daidzein R1 = OH: Genistein

R1 = R2 = OH, R3 = H: Catechins R1 = R2 = R3 = OH: Gallocatechin

R1 = H, R2 = OH: Naringenin R1 = R2 = OH: Eriodictyol R1 = OH, R2 =OCH3: Hesperetin

R1 = H, R2 = H: Pelargonidin R1 = OH, R2 = H: Cyanidin R1 = R2 = OH:Delphinidin R1 = OCH3, R2 = OH: Petunidin R1 = R2 = OCH3: Malvidin

Flavonoids include the flavones (e.g., apigenin, luteolin), flavonols(e.g., quercetin, myricetin), flavonones (e.g., narigenin, hesperidin),flavonols (or catechins) (e.g., epicatechin, gallocatechin),anthocyanidins (e.g., cyaniding, pelargonidin), and isoflavones (e.g.,genistein, daidezin).

Apigenin is a member of the flavone structural class and is chemicallyknown as 4′,5,7,-trihydroxyflavone. Apigenin has the followingstructural formula:

Luteolin is also a member of the flavone structural class and ischemically known as 3′,4′,5,7-tetrahydroxyflavone. Luteolin has thefollowing structural formula):

Both apigenin and luteolin are practically insoluble (i.e., a solubilityof less than 1 mg/ml) in water and nearly all solvents suitable forpharmaceutical, cosmetic, and food additive formulations.

In one embodiment, the invention includes planar ring structured organiccompounds with the proviso that the planar ring structured organiccompound is not a polyphenol, flavonoid and/or ubiquinone Q 10.

The methods of this invention are applicable to poorly solubleflavonoids having a solubility in water less than 1 mg/ml, andparticularly less than 0.1 mg/ml.

II—Methods of Preparing Formulations of the Compounds of the Invention

The subject invention includes multiple ways to formulate planar ringstructured organic compounds allowing a wide variety of applications.Disclosed herein are methods for substantially increasing the solubilityof relatively water insoluble as well as relatively oil insolublecompounds within heat stable solubilizing compounds to enhancedconcentration levels (e. g. up to about 10 or 20 wt % at ambienttemperatures). The solubilized compound can be added to acceptabletopical, subcutaneous, oral, peritoneal, periodontal, aerosol carriersto make formulations.

The subject invention relates to methods for substantially increasingthe solubility concentrations of poorly soluble planar ring structuredorganic compounds, such as a flavonoids, with heat stable non-toxicsolubilizing compounds, such as nonionic surfactant compounds, includingpolysorbates, comprising the steps of:

a) mixing a planar ring structured organic compound, such as aflavonoid, in a heat stable solubilizing compound to form a mixture,

b) heating the mixture while stirring to a temperature where the planarring structured organic compound particulates are solubilized and theresulting mixture (the “concentrate”) forms a clear solution, and

c) cooling the concentrate, and

optionally adding a carrier.

The mixture is heated to an elevated temperature of greater than forexample 100 degrees C., 120 degrees C., 150 degrees C., or 170 degreesC. The temperature selected is that which allows the planar molecules togo into solution. The mixture is heated to a temperature not exceedingthe boiling point or decomposition point of either the planar compoundor the solubilizing compound. The heating step is advantageously donewith only the planar compound and the solubilizing compound present. Thecarrier is advantageously not present during mixing or heating. In manyembodiments, the molar ratio of planar molecule to solubilizing compoundapproaches 1:2. The ratio of planar molecule to solubilizing compoundapproaches 1 mole of planar compound to 2 moles of solubilizingcompound, e.g. surfactant for certain combinations. Significantly moresurfactant than 1 mole active agent to 2 moles of surfactant is requiredfor some active agent/surfactant combinations, e.g. 1 mole active agentto 20 moles of surfactant.

TABLE III Solubility of Various Flavonoids in Surfactants/Solvents viathe Thermal Treatment Process Molar Molar DPSI DPSI DPSI Ratio RatioLit. PS80 ⁽¹⁾Jordi PEG400 ⁽¹⁾Jordi PS80 PEG400 PEG300 PS80/ PEG400/ H2OSol. ~Sol. PS800 Sol. PEG400 Sol. Sol. Sol. AP| AP| MP Sol.- Conc.-Temp. Results Conc. Results Data Data Data (mole/ (mole/ Compound MW °C. (mg/ml) (mg/Ml) (C.) (mg/ml) (mg/ml) (mg/ml) (mg/ml) (mg/ml) (mg/ml)mole) mole) APIGENIN 270 360 0.002 40-60 −270  40.5 34.8 36-45 30-4035-45 4.5 21.7 LUTEOLIN 286 330 0.38 >80 >200 70-90 72.5 3.0 11.4RESVERATROL 228 255 0.1-0.3 >80 >200 127.5 80-100 2.4 QUERCETIN 302 315<1 >80 >200 65 60-80 3.2 HEPERIDEN 610 260 0.05-3   <10: Dec >200 <10:Dec 81 21.2

For creation of a pharmaceutical composition, a nutraceutical, a dietarysupplement, cosmeceutical, a food supplement, or medical food, afterstep b) or c) is the step of adding the solubilized compound mixture toa dermatological, oral, injectable, dermal patch, or aerosol carrier.

In another embodiment is the step of adding after step b) or c) analcohol such as ethyl alcohol to the concentrate to form a solublecompound solution with a reduced viscosity. Other advantageous materialsto reduce the viscosity level of the solubilized compound mixtureinclude: small-chain alcohols (such as isopropyl and benzyl alcohol),ethoxydiglycol (diethylene glycol monoethyl ether or Transcutol),propylene glycol, hexylene glycol, butylene glycol, dipropylene glycol,glycerin, water, saline, DMSO, isopropyl myristate, mineral oil, lowviscosity surfactants, and dimethyl isosorbide.

The method of the subject invention is useful in increasing thesolubility of compounds that have poor solubility or that are currentlysolubilized in such a manner (e.g. as a salt) that precludes or makesdifficult certain applications.

Apigenin/Polysorbate 80 formulations can be made as follows:

-   -   Apigenin powder and viscous liquid polysorbate 80 are mixed in        the ratio from about 5 to 10 wt % of apigenin to 95 to 90 wt %        polysorbate 80. A small quantity (5-10 wt %) of D.I. water and        optionally acetone and/or ethyl alcohol is optionally added to        facilitate the blending of the mixture.    -   This mixture is thoroughly stirred to form a thick paste-like        blend.    -   The mixture is then slowly heated to relatively high        temperatures (about 100 to 150° C.) while stirring. The heating        is accompanied by the boiling off of the water and also volatile        constituents present in the Polysorbate 80.    -   Upon the removal of the volatiles and heating to temperatures in        excess of about 200 to 300° C., a dark brown transparent liquid        results such that all the solid apigenin is solubilized in the        Polysorbate 80 mixture.    -   Upon cooling to ambient temperatures, a thick viscous brown        liquid results. The higher the apigenin content—the darker the        resulting color.    -   Based on a 4.05% concentration of apigenin in the viscous        apigenin polysorbate 80 liquid, the content of apigenin is 40.5        mg/ml or 40, 500 ppm.        It was unanticipated that high temperature levels were necessary        to cause the high solubility level of apigenin and other        relatively water insoluble flavonoids.

The use of apigenin/polysorbate 80 in an alcohol solution can deliverapigenin and other relatively insoluble flavonoids to the desired targetlocation. The invention includes methods of combining heat stablecompounds with the proper balance of polarity characteristics such assurfactants, with other flavonoids to achieve elevated concentrationlevels of the other flavonoids. Examples 2 and 3 show formulations ofother flavonoids and polysorbates.

III. Formulations and Compositions of Planar Ring Compounds

The subject invention relates to compositions comprising a planar ringstructured organic compound, such as a flavonoid, and a heat stablesolubilizing compound such as a surfactant, wherein said composition isformed by mixing the planar compound and the solubilizing compound to anelevated temperature (typically >100° C.) where said planar compound isdissolved in said solubilizing compound. When the dissolved mixture iscooled to ambient temperatures, said dissolved planar compound remainsdissolved even at concentrations exceeding the ambient temperaturesaturation concentration level. The concentration of the planar compoundor active agent in the composition is greater than the ambienttemperature and pressure saturation concentration of the active agent insaid heat stable solubilizing compound, and said composition is not asupersaturated solution. The composition “concentrate’ is stable for atime at least until a formulation is made from the concentrate.

As used herein, “a heat stable solubilizing compound” is a compound thatis stable at least up to the melting of the planar ring structuredorganic compound to be solubilized. Upon thermal treatment (heating),the heat stable solubilizing compound when mixed with a planar ringstructured organic compound, such as a flavonoid, solubilizes the planarring structured organic compound, and upon cooling to ambienttemperatures, continues to solubilize the planar ring structured organiccompound. The mixture is heated to a temperature not exceeding theboiling point or decomposition point of either the planar compound orthe solubilizing compound. For pharmaceutical, nutraceutical food andcosmetic applications the heat stable solubilizing compounds must benon-toxic at the levels used.

Advantageously, the planar ring structured organic compound solubilizingcompound is capable of continuing to solubilize the planar ringstructured organic compound at ambient temperatures for an extendedperiod of time, e.g. 1 or 2 months, advantageously 1 or 2 years, but forat least as long as the time needed for the concentrate to be added to acarrier to make a formulation.

As used herein, “supersaturated solution” is a solution that containshigher than a saturated concentration of a solute; a slight disturbanceor seeding causes crystallization of excess solute.

Heat stable solubilizing compounds that allow for enhanced solubilityconcentration levels of planar cyclic compounds employing the hightemperature methods of this disclosure include sufactants

Surfactants

Surfactants are are classified as follows (see FIG. 1):

Surfactants According to the Composition of their Tail

The tail of surfactants can be:

-   -   A hydrocarbon chain: aromatic hydrocarbons (arenes), alkanes        (alkyl), alkenes, cycloalkanes, alkyne-based;    -   An alkyl ether chain:        -   Ethoxylated surfactants: polyethylene oxides are inserted to            increase the hydrophilic character of a surfactant;        -   Propoxylated surfactants: polypropylene oxides are inserted            to increase the lipophilic character of a surfactant;    -   A fluorocarbon chain: fluorosurfactants;    -   A siloxane chain: siloxane surfactants        A surfactant can have one or two tails, these are called        double-chained.        Surfactants According to the Composition of their Head

Surfactant classification according to the composition of their head:nonionic, anionic, cationic, amphoteric.

A surfactant can be classified by the presence of formally chargedgroups in its head. A non-ionic surfactant has no charge groups in itshead. The head of an ionic surfactant carries a net charge. If thecharge is negative, the surfactant is more specifically called anionic;if the charge is positive, it is called cationic. If a surfactantcontains a head with two oppositely charged groups, it is termedzwitterionic.

Some commonly encountered surfactants of each type include:

-   -   Ionic        -   Anionic: based on permanent anions (sulfate, sulfonate,            phosphate) or pH-dependent anions (carboxylate):            -   Sulfates:                -   Alkyl sulfates: ammonium lauryl sulfate, sodium                    lauryl sulfate (SDS, sodium dodecyl sulfate, another                    name for the compound);                -   Alkyl ether sulfates: sodium laureth sulfate, also                    known as sodium lauryl ether sulfate (SLES), sodium                    myreth sulfate;            -   Sulfonates:                -   Docusates: dioctyl sodium sulfosuccinate;                -   Sulfonate fluorosurfactants:                    perfluorooctanesulfonate (PFOS),                    perfluorobutanesulfonate;                -   Alkyl benzene sulfonates;            -   Phosphates:                -   Alkyl aryl ether phosphate                -   Alkyl ether phosphate            -   Carboxylates:    -   Alkyl carboxylates: Fatty acid salts (soaps): sodium stearate;    -   Sodium lauroyl sarcosinate;    -   Carboxylate fluorosurfactants: perfluorononanoate,        perfluorooctanoate (PFOA or PFO)        -   Cationic: based on:            -   pH-dependent primary, secondary or tertiary amines:                primary amines become positively charged at pH<10,                secondary amines become charged at pH<4:                -   Octenidine dihydrochloride;            -   Permanently charged quaternary ammonium cation:                -   Alkyltrimethylammonium salts: cetyl                    trimethylammonium bromide                -   (CTAB) a.k.a. hexadecyl trimethyl ammonium bromide,                    cetyl trimethylammonium chloride (CTAC);                -   Cetylpyridinium chloride (CPC);                -   Polyethoxylated tallow amine (POEA);                -   Benzalkonium chloride (BAC);                -   Benzethonium chloride (BZT);                -   5-Bromo-5-nitro-1,3-dioxane;                -   Dimethyldioctadecylammonium chloride                -   Dioctadecyldimethylammonium bromide (DODAB)        -   Zwitterionic (amphoteric): based on primary, secondary or            tertiary amines or quaternary ammonium cation with:            -   Sulfonates:                -   CHAPS                    (3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate);                -   Sultaines: cocamidopropyl hydroxysultaine;            -   Carboxylates:                -   Amino acids                -   Imino acids                -   Betaines: cocamidopropyl betaine;            -   Phosphates: lecithin    -   Nonionic        -   Fatty alcohols:            -   Cetyl alcohol,            -   Stearyl alcohol,            -   Cetostearyl alcohol (consisting predominantly of cetyl                and stearyl alcohols),            -   Oleyl alcohol;        -   Polyoxyethylene glycol alkyl ethers (Brij):            CH₃—(CH₂)₁₀₋₁₆—(O—C₂H₄)₁₋₂₅—OH:            -   Octaethylene glycol monododecyl ether,            -   Pentaethylene glycol monododecyl ether;        -   Polyoxypropylene glycol alkyl ethers:            CH₃—(CH₂)₁₀₋₁₆—(O—C₃H₆)₁₋₂₅—OH;        -   Glucoside alkyl ethers: CH₃—(CH₂)₁₀₋₁₆—(O-Glucoside)₁₋₃-OH:            -   Decyl glucoside,            -   Lauryl glucoside,            -   Octyl glucoside;        -   Polyoxyethylene glycol octylphenol ethers:            C₈H₁₇—(C₆H₄)—(O—C₂H₄)₁₋₂₅—OH:            -   Triton X-100;        -   Polyoxyethylene glycol alkylphenol ethers:            C₉H₁₉—(C₆H₄)—(O—C₂H₄)₁₋₂₅—OH:            -   Nonoxynol-9;        -   Glycerol alkyl esters:            -   Glyceryllaurate        -   Polyoxyethylene glycol sorbitan alkyl esters: Polysorbates;        -   Sorbitan alkyl esters: Spans;        -   Cocamide MEA, cocamide DEA;        -   Dodecyldimethylamine oxide;        -   Block copolymers of polyethylene glycol and polypropylene            glycol: Poloxamers            Surfactants According to the Composition of their            Counter-Ion

In the case of ionic surfactants, the counter-ion can be:

-   -   Monoatomic/Inorganic:        -   Cations: metals: alkali metal, alkaline earth metal,            transition metal;        -   Anions: halides: chloride (Cl⁻), bromide (Br⁻), iodide (I⁻);    -   Polyatomic/Organic:        -   Cations: ammonium, pyridinium, triethanolamine (TEA)        -   Anions: tosyls, trifluoromethanesulfonates, methylsulfate            Advantageous Surfactants are as Follows:

Amphoteric Surfactants

ALKYL AMMONIUM SULFONIC ACID

BETAINE

AMPHOTERIC-2

AMPHOTERIC-9

DISODIUM COCOAMPHODIACETATE

PHOSPHOLIPID

-   -   USES/FUNCTIONS: Cleansers, Solubilizers, Emulsifiers    -   ADMINISTRATION ROUTE: Topical, IV (Infusion)

Anionic Surfactants

ALKYL ARYL SODIUM SULFONATE

AMMONIUM LAURYL SULFATE

AMMONIUM NONOXYNOL-4 SULFATE

DISODIUM LAURETH SULFOSUCCINATE

DOCUSATE SODIUM/SODIUM BENZOATE

GLYCERYL STEARATE SE

LAURYL SULFATE

SODIUM CETOSTEARYL SULFATE

SODIUM DODECYLBENZENESULFONATE

SODIUM SULFOSUCCINATED UNDECYCLENIC

MONOALKYLOLAMIDE

TROLAMINE LAURYL SULFATE

-   -   USES/FUNCTIONS: Cleansers, Wetting agents, Solubilizers    -   ADMINISTRATION ROUTE: Topical, Oral, Respiratory (inhalation),        Sublingual

Cationic Surfactants

ALUMINUM STEARATE

APRICOT KERNEL OIL PEG-6 ESTERS

BEHENETH-10

-   -   USES/FUNCTIONS: Emulsifiers, Solubilizers, Preservatives    -   ADMINISTRATION ROUTE: Topical, Oral Ophthalmic, Nasal,        Intramuscular, Auricular

Nonionic Surfactants

BENZALKONIUM CHLORIDE

CETEARETH-12, -15, -30

CETEARYL ALCOHOL/CETEARETH-20

CETETH-2, -10, -20

GLYCERYL STEARATE-LAURETH-23

GLYCERYL STEARATE/PEG STEARATE

GLYCERYL STEARATE/PEG-100 STEARATE

GLYCERYL STEARATE/PEG-40 STEARATE

LANOLIN NONIONIC DERIVATIVES

LANOLIN, ETHOXYLATED

LAURETH-2, -4, -23

LAUROYL POLYOXYLGLYCERIDES

OCTOXYNOL 9, -40

PEG VEGETABLE OIL

PEG-120 METHYL GLUCOSE DIOLEATE

PEG-150 DISTEARATE

PEG-22 METHYL ETHER/DODECYL

GLYCOL COPOLYMER

PEG-25 PROPYLENE GLYCOL STEARATE

PEG-75 LANOLIN

PEG-8 CAPRYLIC/CAPRIC GLYCERIDES

PEG-8 LAURATE

PEGLICOL-5-OLEATE

PEGOXOL 7 STEARATE

POLOXAMER 124, −181, −182, −188, −237, −331, −338, −407

POLYGLYCERYL-10 OLEATE

POLYGLYCERYL-10 TETRALINOLEATE

POLYGLYCERYL-3 OLEATE, -4 OLEATE

POLYOXYL 100 GLYCERYL STEARATE

POLYOXYL 12 GLYCERYL LAURATE

POLYOXYL 2 STEARATE

POLYOXYL 20 CETOSTEARYL ETHER

POLYOXYL 20 STEARATE

POLYOXYL 35 CASTOR OIL

POLYOXYL 4 LAURATE

POLYOXYL 40 CASTOR OIL

POLYOXYL 40 HYDROGENATED CASTOR OIL

POLYOXYL 40 STEARATE, -50 STEARATE, -400 STEARATE

POLYOXYL 6 AND POLYOXYL 32

PALMITOSTEARATE

POLYOXYL 6 ISOSTEARATE

POLYOXYL 60 CASTOR OIL

POLYOXYL 60 HYDROGENATED CASTOR OIL

POLYOXYL 8 STEARATE

POLYOXYL GLYCERYL STEARATE

POLYOXYL LANOLIN, PALMITATE, -STEARATE

POLYSORBATE 20, -40, -60, -65, -80

PPG-15 STEARYL ETHER

PROPYLENE GLYCOL MONOSTEARATE

SORBITAN MONOLAURATE,

SORBITAN MONOPALMITATE

SORBITAN MONOSTEARATE

SORBITAN TRIOLEATE

STEARAMIDOETHYL DIETHYLAMINE

STEARETH-2, -10, -20, -21, -40, -100

SUCROSE PALMITATE

SUCROSESTEARATE

WAX, EMULSIFYING

-   -   ADMINISTRATION ROUTE Topical, Oral, Intramuscular,        Intralesional, Auricular (OTIC), Ophthalmic, Transdermal,        Subcutaneous, IV (Infusion), Nasal, Periodontal, Vaginal    -   USES/FUNCTIONS Emulsifiers, Solubilizers, Wetting agents,        Gelling agents

Silicone-Based Surfactants

Also known as organosilicones, these are increasing in popularitybecause of their superior spreading ability. This class contains apolysiloxane chain. Some of these are a blend of non-ionic surfactants(NIS) and silicone while others are entirely silicone.

Non-Ionic Surfactants

Fatty acid esters of sorbitan (generally referred to as spans) and theirethoxylated derivatives (generally referred to as polysorbates) areperhaps the most commonly used nonionics. They can be used alone or incombination (e.g. polysorbate 80 and span 80) to form mixed micelles.The sorbitan esters are insoluble in water, but soluble in most organicsolvents (low Hydrophile-Lipophile Balance (HLB) number surfactants).The ethoxylated products are generally soluble in water and haverelatively high HLB numbers. These nonionic surfactants can be usedalone or in a suitable combination to form mixed micelles of the desiredHLB. One of the main advantages of the sorbitan esters and theirethoxylated derivatives is their approval as food additives. They arealso used in cosmetics and pharmaceutical preparations.

Nonionic surfactant compounds that are useful for enhanced solubilityconcentration levels of flavonoids having solubility in water less than1 mg/ml in water (employing the thermal treatment methods of thisinvention) include: ethoxylated aliphatic alcohols; polyoxyethylenesurfactants; carboxylic esters; polyethylene glycol esters;anhydrosorbitol ester and its ethoxylated derivatives; glycol esters offatty acids; and fatty amine ethoxylates.

The most common nonionic surfactants are those based on ethylene oxide,referred to as ethoxylated surfactants. Several classes can bedistinguished: alcohol ethoxylates, alkyl phenol ethoxylates, fatty acidethoxylates, monoalkaolamide ethoxylates, sorbitan ester and theirethoxylated derivates, ethoxylates, fatty amine ethoxylates, andethylene oxide-propylene oxide copolymers (sometimes referred to aspolymeric surfactants). Another important class of nonionics is themultihydroxy products such as glycol esters, glycerol (and polyglycerol)esters, glucosides (and polyglucosides) and sucrose esters. Amine oxidesand sulphinyl surfactants represent nonionics with a small head group.(M J Schick (ed.): Nonionic Surfactants: Physical Chemistry, MarcelDekker, New York, 1987)

HLB is an empirical expression for the relationship of the hydrophilic(“water-loving”) and hydrophobic (“water-hating”) groups of asurfactant. The higher the HLB value, the more water-soluble is thesurfactant. The most common emulsion type, oil-in-water (o/w), oftenrequires higher HLB surfactants—preferably 12-16 while water-in-oilemulsions (w/o) require low HLB surfactants—preferable 7-11. Surfactantswith an HLB value<10 are oil-soluble while those >10 are water-soluble.

Span 20 is very suitable for water-in-oil topical formulations whilePolysorbate 80 is very appropriate for solubilizing compounds inoil-in-water topical formulations. Surfactants with high HLB values likePolysorbate 80 are also applicable for making “concentrates” intendedfor use in the preparation of hydrophilic aqueous-based formulations andincorporation into to the aqueous phase of an emulsion. Whereas,surfactants with low HLB values like Span 20 are also applicable formaking “concentrates” intended for use in the preparation of lipophilicnonaqueous-based formulations and incorporation into to the non-aqueousor oil phase of an emulsion.

Polysorbate Surfactants

Polysorbates (commercially also known as Tweens) are nonionicsurfactants and emulsifiers derived from polyethoxylated sorbitan andfatty acids. They are often used in foods and in cosmetics to solubilizeessential oils into water-based products. The polysorbates are viscous,water-soluble pale yellow liquids. Polysorbates also help to formemulsions by reducing the surface tension of the substances to beemulsified. Polysorbates have been recognized for their ability to helpingredients to dissolve in a solvent in which they would not normallydissolve. Polysorbates function to disperse oil in water as opposed towater in oil.

Polysorbates are produced by reacting the polyol, sorbitol, withethylene oxide. The polyoxyethylenated sorbitan is then reacted withfatty acids obtained from vegetable fats and oils such as stearic acid,lauric acid, and oleic acid. Surfactants that are esters of plain(non-PEG-ylated) sorbitan with fatty acids are usually referred to bythe name Span.

The polysorbates are composed of fatty acid esters of polyoxyethylenesorbitan, and their structures are typically presented as the chemicallyhomogenous polysorbates shown in below. While the number of repeatethylene oxide subunits varies at each position, their total number(w+x+y+z) is constant for each polysorbate (i.e., 20, 40 60, 80).

Polysorbates are a class of emulsifiers used in some pharmaceuticals andfood preparation. They are often used in cosmetics to solubilizeessential oils into water-based products. Polysorbates are oily liquidsderived from PEG-ylated sorbitan (a derivative of sorbitol) esterifiedwith fatty acids. Surfactants that are esters of plain (non-PEG-ylated)sorbitan with fatty acids are usually referred to by the name Span.

-   -   Polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate)    -   Polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate)    -   Polysorbate 60 (polyoxyethylene (20) sorbitan monostearate)    -   Polysorbate 80 (polyoxyethylene (20) sorbitan monooleate)        The number 20 following the polyoxyethylene part refers to the        total number of oxyethylene —(CH₂CH₂)— groups found in the        molecule. The number following the polysorbate part is related        to the type of fatty acid associated with the polyoxyethylene        sorbitan part of the molecule. Monolaurate is indicated by 20,        monopalmitate is indicated by 60, and monooleate by 80. The same        numbering is followed in their Span equivalents (Span 20, Span        40, Span 60 and Span 80).

The invention includes methods for increasing the solubilityconcentrations of poorly soluble compounds with polysorbates. Aspreviously noted, many flavonoids and specifically apigenin are poorlysoluble in aqueous solutions thus severely limiting theirbioavailability for topical, pharmaceutical and nutraceuticalapplications.

The subject invention includes a method for increasing the aqueous phasesolubility levels of polyphenols by utilizing surfactant compounds, inparticular Polysorbates 80, 60, 40 and 20. It should be noted that inthis example polysorbates 20, 40 and 60 represent a homologous series ofpolysorbates with varying saturated fatty acids. The number of carbonsin the fatty acid chain increases from 12 (polysorbate 20) to 18(Polysorbate 60). Polysorbate 80 represents an unsaturated fatty acidwith 18 carbon chain length (Oleate). These examples are not allinclusive and one trained in the art should recognize the usefulness ofthese types of nonionic surfactants with any other fatty acid and alsoother nonionic surfactants of other classes such as polyoxyethylenealkyl ethers of fatty acids.

PEG

Poly(ethylene glycol) (PEG), otherwise known as poly(oxyethylene) orpoly(ethylene oxide) (PEO), is a synthetic polyether that is readilyavailable in a range of molecular weights (MW). Materials withMW<100,000 are usually called PEGs, while higher molecular weightpolymers are classified as PEOs. These polymers are amphiphilic andsoluble in water as well as in many organic solvents. Low molecularweight (MW<1,000) PEGs are viscous and colorless liquids, while highermolecular weight PEGs are waxy, white solids with melting pointsproportional to their molecular weights to an upper limit of about 67°C. PEG or PEO has the following structure,

The numbers that are often included in the names of PEGs indicate theiraverage molecular weights, e.g., a PEG with n=9 would have an averagemolecular weight of approximately 400 daltons and would be labeled PEG400.

Anionic and Cationic Surfactants

It should also be noted that either anionic surfactants such asdocussate sodium or sodium lauryl sulfate or cationic surfactants suchas cetrimide or benzethonium chloride can also be used either alone orin combination with nonionic surfactants in the formulations of theinvention. Significant aqueous phase enhancements exceeding more thantwo orders of magnitude have been achieved for several relatively waterinsoluble polyphenols.

The novel formulations with high planar compound concentrations can beutilized in oral, inhalation, topical, peritoneal, periodontal andsuppository formulations. They are useful in pharmaceutical,cosmeceutical and nutraceutical applications (see below).

As used herein, the term “pharmaceutical composition” or “pharmaceuticalformulation” shall mean a composition wherein the components of thecomposition or formulation are of pharmaceutical grade. The compositionsor formulations can conveniently be presented in unit dosage form, andcan be prepared by methods known in the art of pharmacy. Theformulations can be for immediate, or slow or controlled release of thediffusion enhancing compound. The advantages of a sustained releasesystem (also known as time release, controlled release, etc.) are thatdosing frequency can decrease and the systemic drug concentrations aresteadier for a longer duration as compared to other formulations of thesame drug. Appropriate dosages of the compositions of the invention willdepend on the mode of administration, metabolism of the given compound,and the severity of the condition being treated.

The subject invention includes multiple ways to formulate planarcompounds such as flavonoids, allowing a wide variety of applications.The compounds, compositions and formulations of the invention are usefulin the prevention of and the treatment of the disorders and diseasesdiscussed below. As used herein, a “therapeutically effective amount” isthe dose necessary to have the desired effect. For example in the caseof plaque psoriasis, a therapeutically effective amount is that amountwhich reduces the sizes or severity of the patches or plaques. A“prophylactic amount” is that dose which prevents or reduces thelikelihood of a disorder or disease occurring.

Table IV lists a variety of dosage types and forms that can serve as ameans for delivering the subject formulations.

TABLE IV DOSAGE TYPES & FORMS TYPE FORMS ORAL Capsule, Thin film, LiquidSolutions INHALATION Aerosol, Inhaler, Nebulizer, Smoking, VaporizerPERIDONTAL Liquid Solution, Paste, Spray PARENTERAL Intradermal,Intramuscular, Intraosseous, INJECTION Intraperitoneal, Intravenous,Subcutaneous TOPICAL Cream, Gel, Liniment or Balm, Lotion, Ointment,Solution, Spray, Foam, Ear drops, Eye drops, Skin patch (transdermal)SUPPOSITORY Rectal (e.g., enema), Vaginal (e.g., douche, pessary, etc.)A. Topical Administration

Topical administration of solubilized compounds is typically done in theform of a patch, lotion, cream, gel, solution, spray, liquids andserums, foam or ointment. The methods described above for increasing thesolubility levels of flavonoids or other poorly soluble planar compoundsin surfactant enable the formulation of lotion, cream, gel, solution,spray, foam or ointment topical products wherein the planar compound isin solution; a physical state of the relatively insoluble planarcompound not achievable without the use of these methods.

Transdermal Delivery

The methods described for increasing the solubility levels of planarcompounds within surfactants enable the transdermal delivery into thesystemic circulation via permeation at a controlled rate. The subjectformulations offer a noninvasive route of drug administration byaddressing issues related to the inherently low permeability of skin.The skin is a good barrier to drug penetration. Incorporation ofpenetration enhancers facilitates the absorption of drugs by alteringthe barrier property of the stratum corneum. Several nonionicsurfactants such as polysorbate 80 in topical, oral, and peritonealapplications are considered to be pharmacologically inert, nontoxic,nonirritating, nonallergic, odorless, compatible with most drug andexcipients, and have good solvent properties.

Penetration Enhancers

Different classes of penetration enhancers including alcohols andpolyols (ethanol, glycerol, propylene glycol), surfactants (Tween,Span), fatty acids (Oleic acid), amines and amides (Azone,N-methylpyrrolidone), terpenes (limonene) sulfoxides(dimethylsulfoxide-DMSO), esters (isopropyl myristate) have beendeveloped over the past two decades (French E, Potton C, Walters K.Pharmaceutical skin penetration enhancement. In: Walters K, Hadgraft J,editors. New York: Marcel Dekker; 1993. p. 113-44).

Microemulsions

Another formulation approach aiming to enhance skin penetration is thepreparation of microemulsions. Microemulsions consist of water, oil, andsurfactant that yield a transparent thermodynamically stable liquid.Properties of microemulsions include optical transparency, thermodynamicstability, and solubility of both hydrophobic and hydrophiliccomponents. Microemulsions are clear, stable, isotropic liquid mixturesof oil, water and surfactant, frequently in combination with acosurfactant. The aqueous phase may contain salt(s) and/or otheringredients, and the “oil” may actually be a complex mixture ofdifferent hydrocarbons and olefins. In contrast to ordinary emulsions,microemulsions form upon simple mixing of the components and do notrequire the high shear conditions generally used in the formation ofordinary emulsions. The two basic types of microemulsions are direct(oil dispersed in water, o/w) and reversed (water dispersed in oil,w/o).

Penetration enhancement from microemulsions can be due to an increase indrug concentration which provides a large concentration gradient fromthe vehicle to the skin. The nonionic surfactants solvents containingthe enhanced flavonoid concentrations (described herein) are well suitedfor the preparation of microemulsions for transdermal, oral andperitoneal applications.

In one embodiment, a microemulsion contains apigenin is dissolved inpolysorbate 80 together with water and ethyl alcohol as a cosurfactantand an oil phase of isoproyl myristate (IPM). This embodiment hastopical applications, due to skin penetration properties, as well asoral, injection and nasal spray applications.

The formulations disclosed in this invention allow enhanced transdermaldrug delivery methodologies for flavonoids. Of particulate note are thedisclosed formulations of relatively water insoluble flavonoids,including apigenin, solubilized in nonionic surfactants mixtures. Invitro skin penetration testing with human and mouse skins demonstratedunexpectedly high apigenin accumulation within the epidermal and dermallayers resulting from the application of the disclosed nonionicsurfactant mixtures. See Example 15.

Transdermal Patches

Useful for transdermal active agent delivery of poorly soluble planarcompounds, is the use of transdermal patches containing the solubilizedcompound within the solubilizing agent such as a surfactant, dilutedwith an alcohol such as the relatively volatile ethyl alcohol. The outernonporous barrier of the patch when applied to the skin serves to reducethe evaporation of the relatively volatile alcohol thereby allowing forthe increased penetration and delivery of the active agent. Othersolvent diluents used in cosmetic and foods applications such asalcohols (i.e., ethyl alcohol, glycols, ethoxydiglycol etc.), esters(dimethyl isosorbide etc.) serve to reduced the viscosities ofrelatively viscous nonionic surfactant thereby increasing the rate anddepth of skin penetration when applied to the skin's surface orcontained within transdermal patches. Particularly, useful for dermalpatch and transdermal patch, are the use of microemulsion formulationsof active agents. The formulations consist of oil-in-water andwater-in-oil type microemulsions.

Transdermal patches can be classified into two types of deliverysystems—reservoir based and matrix based. Compositions for both aresimilar except that semipermeable membranes are used to control thediffusion from the reservoir system. Examples of membranes used includepolypropylene, low density polyethylene, ethylene-vinyl acetateco-polymer etc. In matrix based formulations drugs can bedispersed/solubilized in the adhesives. Two commonly used adhesiveclasses include acrylate and silicone based materials. Examples ofpressure sensitive acrylate adhesives include, but are not limited to,the DURO-TAK® series (Henkel, USA). Examples of pressure sensitivesilicone adhesives include, but are not limited to, the Bio-PSA® series(Dow Corning, USA). Additional information relating to some specificacrylate and silicone based pressure sensitive adhesives are summarizedin Table V.

TABLE V A SUMMARY OF ACRYATE & SILICONE BASED PRESSURE SENSITIVEADHESIVES ADHESIVE SOLVENT DESCRIPTION SYSTEM POLYMER NOTES DURO-TAK ®Ethyl Acrylic Reactive or 87-900A acetate non-curing sensitive API'sDURO-TAK ® Ethyl Acrylate- Long term (3)87-2516 acetate vinylacetate;wear Ethanol self-curing DURO-TAK ® Ethyl Acrylate- Long term 87-4287acetate vinylacetate; wear non-curing BIO-PSA 7-4202 EthylTrimethylsiloxy Amine- Silicone adhesive acetate silanol endblockedcompatible BIO-PSA 7-4302 PDMS Silicone adhesive

Solvents and penetration enhancers known to those skilled in the art canalso be included in the compositions. Potential, solvents/enhancers caninclude but are not limited to fatty acids (oleic acid), esters(isopropyl myristate), alcohols (ethyl and isopropyl) and glycols(propylene glycol, hexylene glycol). Other components can includeantioxidants (e.g. BHT and BHA) or chelating agents (e.g. citric acid).

In Example 15, the formulations of the subject invention deliveredsignificant apigenin concentrations to both the epidermal and dermalskin layers.

B. Oral Administration

Formulations of this invention can also be administered orally. For oraladministration, compositions disclosed herein can be in the form of, forexample, liquid gel capsules or solutions. For oral administration, thecompositions disclosed can be in any orally acceptable dosage formincluding, but not limited to emulsions, microemulsions, and aqueoussolutions, and liquid gel capsules.

When the compounds are prepared for oral administration, they aregenerally combined with a pharmaceutically acceptable carrier, diluentor excipient to form a pharmaceutical formulation, or unit dosage form.For oral administration, compounds can be present as a solution, asuspension, an emulsion or in a natural or synthetic polymer or resinfor ingestion of the active ingredients from a chewing gum.

Orally administered compounds can also be formulated for sustainedrelease, e. g., flavonoids can be coated, micro-encapsulated, orotherwise placed within a sustained delivery device. The total activeingredients in such formulations comprise from 0.01 to 10% by weight ofthe formulation.

Pharmaceutical formulations can be prepared by procedures known in theart using well-known and readily available ingredients. For example, theflavonoids can be formulated with common excipients, diluents, orcarriers, and formed into dosage forms such as capsules, solutions,suspensions, aerosols and the like. All of these dosage forms can be forimmediate release, sustained release or enteric coated. These can beeither for peroral or sublingual or buccal delivery. Examples ofexcipients, diluents, and carriers that are suitable for suchformulations include fillers and extenders such as starch, cellulose,sugars, mannitol, and silicic derivatives. Binding agents can also beincluded such as carboxymethyl cellulose, hydroxymethylcellulose,hydroxypropyl methylcellulose and other cellulose derivatives,alginates, gelatin, and polyvinyl-pyrrolidone. Moisturizing agents canbe included such as glycerol, disintegrating agents such as calciumcarbonate and sodium bicarbonate. Agents for retarding dissolution canalso be included such as paraffin. Resorption accelerators such asquaternary ammonium compounds can also be included. Surface activeagents such as cetyl alcohol and glycerol monostearate can be included.Adsorptive carriers such as kaolin and bentonite can be added.Lubricants such as talc, calcium and magnesium stearate, and solidpolyethyl glycols can also be included.

The compositions of the invention can also contain thickening agentssuch as cellulose and/or cellulose derivatives. They can also containgums such as xanthan, guar gum or gum arabic, or alternativelypolyethylene glycols, bentones and the like.

Soft gelatin capsules containing flavonoids can contain inactiveingredients such as gelatin, microcrystalline cellulose, glycerin,sodium lauryl sulfate, starch, talc, and titanium dioxide, and the like,as well as liquid vehicles such as polyethylene glycols (PEGs) andvegetable oil. Moreover, enteric-coated capsules containing flavonoidsor other compounds are designed to resist disintegration in the stomachand dissolve in the more neutral to alkaline environment of the duodenumare typically coated with cellulose acetate derivatives.

The planar compounds can also be formulated as elixirs or solutions forconvenient oral administration. The pharmaceutical formulations of theflavonoids can also take the form of an aqueous or anhydrous solution ordispersion, or alternatively the form of an emulsion or suspension.

C. Parenteral Administration

The formulations of this invention can also be administered parentally.For parenteral administration, the compositions disclosed herein can bein the form of injectable solutions or suspensions, such as salinesolutions. The term “parenteral,” as used herein includes intravenous,subcutaneous, intramuscular, intrasynovial, intrasternal, intralesionaland intracranial injection or infusion techniques. Typical formulationsinclude emulsions and microemulsions. Injectable formulations, includingemulsions, frequently consist of mixtures of purified water forinjection, organic cosolvents, surfactants, suspending agents,preservatives, antioxidants and pH adjusters. Examples of ingredientsillustrating each category are as follows, but not limited to:

Cosolvents

Propylene glycol, ethyl alcohol, glycerin, polyethylene glycols, benzylalcohol, vegetable oil, soybean oil, safflower oil, cottonseed oil, cornoil, peanut oil, sunflower oil, arachis oil, castor oil, olive oil,ester of a medium or long chain fatty acid such as a mono- di- ortriglyceride, ethyl oleate, isopropyl myristate, polyoxyl hydrogenatedcastor oil, phospholipids and combinations thereof.

Surfactants

Polyoxyethylene/polyoxypropylene block copolymers, phosphatides, andpolysorbates are commonly used as synthetic nonionic surfactants

Suspending Agents

Polyvinyl pyrrolidone (PVP), sodium carboxymethylcellulose and dextran

Preservatives

Disodium edetate, sodium benzoate, benzalkonium chloride, benzoic acidmethylparaben and propylparaben

Antioxidants

Ascorbic acid, butylated hydroxytoluene, butylated hydroxyanisole,sodium thiosulfate

pH Adjusters

Sodium hydroxide, tromethamine, sodium citrate, sodium phosphate dibasicand monobasic, sodium acetate, citric acid, phosphoric acid, acetic acidand phosphoric acid.

D. Inhalation Administration

The formulations of this invention can also be administered byinhalation means. For inhalation administration, the compositionsdisclosed herein can be in the form of aerosols which deliver theflavonoid ingredients as a suspension of fine liquid droplets in a gasto the mouth or nasal passages. Vaporizer and inhalation devicesfacilitate in the delivery of the flavonoid ingredients.

The compositions can be administered to the respiratory tract. Thecomposition can be presented in unit dosage form in, for example,capsules or cartridges, or, e.g., gelatin or blister packs from whichthe composition may be administered with the aid of an inhalator,insufflator, or a metered-dose inhaler (MDI) or dry powder inhaler(DPI).

The compounds can also be administered in an aqueous solution whenadministered in an aerosol or with a dropper. Thus, other aerosolpharmaceutical formulations can comprise, for example, a physiologicallyacceptable buffered saline solution containing between about 0.01-10% ofthe disclosed flavonoid ingredients. Liquid formulations may alsocontain preservatives such methyl and propyl paraben, benzalkoniumchloride etc, buffers such as phosphate and citrate buffers, tonicityadjusters such as mannitol, sodium chloride etc and antioxidants such asascorbic acid, sodium metabisulfite, sodium thiosulfate etc and colorssuch as D&C yellow #10, FD&C yellow #6 etc.

For administration to the upper (nasal) or lower respiratory tract byinhalation, the active agents are conveniently delivered from anebulizer or a pressurized pack or other convenient means of deliveringan aerosol spray. Pressurized packs can comprise a suitable propellantsuch as dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit can be determined byproviding a valve to deliver a metered amount. Products can also bedelivered by use of nebulizers.

For intra-nasal administration, the therapeutic agent can also beadministered via nose drops, a liquid spray, such as via a plasticbottle atomizer or metered-dose inhaler. Typical of atomizers are theMistometer (Wintrop) and the Medihaler (Riker).

Carriers and Vehicles

In addition to the active agents, the formulations comprise one or morevehicle such as a “pharmaceutically acceptable” or cosmetically or“dermatologically acceptable” carrier/vehicle. A “pharmaceuticallyacceptable carrier” does not substantially adversely affect thepharmacological activities of the active agent, is not deleterious orunsuitably harmful to the recipient thereof and is non-toxic whenadministered at dosages sufficient to deliver an effective amount of theactive ingredient, and the carrier (diluent, excipient, and/or saltetc.) is compatible with the other ingredients of the formulation.Likewise, a “dermatologically acceptable carrier” has the samequalities.

A dermatologically acceptable carrier typically includes ingredientsthat are chemically and physically compatible with the activeingredient(s), stable with an adequate shelf life, and that aid indelivery of the active ingredient(s) into the skin (e.g., to theepidermis and/or dermis) following topical administration. Optionally,the dermatological carrier contains ingredients that contribute to theease of application and have pleasing aesthetic properties (color,scent, feel etc.).

Formulation objectives with respect to the drug delivery profile dependon the intended use of a topical product. For sunscreens, antifungals,and keratolytic formulations, enhanced drug delivery and retention inthe stratum corneum (the outer layer of skin) is desired. Conversely,topical formulations that are intended to modify the physiology of theskin require drug deposition in and often through the lower layers ofthe skin (viable epidermis and dermis).

The carrier can act, for example, as a diluent, dispersant, and/orcarrier for other materials present in the formulation (for example, soas to facilitate their distribution when the composition is applied tothe skin). Some exemplary vehicles include: organic constituents (suchas alcohols, oils, and the like), aqueous based solvents (e.g., thosewhich can dissolve or disperse the active flavone ingredients, e.g., atconcentrations that are suitable for use in the therapeutic treatment).

More specifically, the carrier(s) can include ethanol, isopropanol,benzyl alcohol, glycol (e.g., polyethylene glycols, propylene glycol,ethoxydiglycol, and so forth), oils (such as grapeseed, jojoba, coconut,sesame, mineral etc.), glycerol, fatty acid esters, dimethyl isosorbide,as well as combinations comprising at least one of the foregoingcarriers.

The carrier, which can be present in the formulation in an amount ofless than or equal to 99.99 wt %, for example, 80 wt % to 99.99 wt %,based upon a total weight of the formulation, can be in any of thevarious forms of the desired final formulation as discussed above.

Carrier components in addition to water and oils can also include liquidemollients, solid emollients, solvents, humectants, thickeners, powders,fragrances, odor masking agents, colorants, dispersants, lubricants,silicates as well as combinations comprising at least one of theforegoing. Exemplary solvents include ethyl alcohol, isopropanol,ethoxydiglycol, and dimethyl isosorbide, and acetone, as the preventionand/or relief of dryness, and/or for the protection of the skin, such asstearyl alcohol, cetyl alcohol, acetylated lanolin alcohols, stearicacid, isobutyl palmitate, isocetyl stearate, cetyl palmitate, isopropylstearate, butyl stearate, lanolin, cocoa butter, shea butter, oil (e.g.,olive oil, sunflower seed oil, avocado oil, mineral oil), petroleumjelly, and myristate (e.g., butyl myristate, isopropyl myristate,myristyl myristate), as well as combinations comprising at least one ofthe foregoing.

In an embodiment of the invention, the compositions are formulated withan enteric coating to release the active agent in the intestines.

Additives

Hyaluronic Acid (HA)

Within the dermal structure, HA functions as a space filling, structurestabilizing, and cell protective molecule with remarkable malleablephysical and superb biocompatibility properties. Additionally, HAstructures, which have a high level of visoelasticity, serve to preservea high level of hydration with this skin. A strong correlation existsbetween the water content in the skin and levels of HA within the dermaltissue. It is well documented that there are significant alterations inHA physical and biological properties as skin ages—particularly inmetabolism, content and deterioration in the mechanical properties ofthe skin. It is believed that the maintaining of a viable HA presencewithin the skin's intercellular structure contributes to the viabilityof a healthy skin physical appearance.

In another aspect, it has been well documented that polysaccharidemolecules such as HA do degrade as a consequence of enzymatic andoxidative (free radical) mechanisms. Consequently, it is desirable todevelop topical formulations that serve to prevent the decomposition ofpolysaccharides such as HA. To this end, flavonoids such as flavonesserve to meet this need via their well-documented anti-hyaludonidase andanti-oxidant properties—thereby serving to maintain the viability of HAdesirable functions protecting against the mechanisms which contributeto its breakdown.

Topically, HA has water storing properties, making it beneficial as aswelling agent and lubricant, enabling its incorporation into cosmeticsleading to a perceptible and visible improvement of skin condition. Inuse, it forms a thin transparent visco elastic surface film that helpsto preserve the characteristics of youthful and healthy skin:suppleness, elasticity and tone. Increased skin hydration may swell andopen up the compact structure of the stratum corneum, leading to anincrease in penetration of the active flavonoids ingredients of thetopical formulations described herein.

The formulation can further comprise additive(s) so long as the specificadditive(s) do not adversely affect the active ingredient(s). Somepossible additive(s) that can be used in the various embodiments of theformulation include:

antioxidant(s) (e.g., tocopherol, tocopheryl acetate, butylatedhydroxytoluene, sodium metabisulfite, sodium thiosulfate, and propylgallate),

surfactant(s) (e.g., that can reduce the interfacial tension betweenphases and/or improve stability of the formulation, and/or that can actas emulsifiers, such as glyceryl stearate, stearyl alcohol, cetylalcohol, stearic acid dimethicone, a silicone (siloxane) surfactant,polysorbates, sodium laureth),

skin conditioning agent(s) such as silicone oils,

preservative(s) (e.g., methylparaben, propylparaben, benzyl alcohol,benzalkonium chloride etc.),

humectants(s) or emollients or moisturizers such as glycerol,polyethylene glycol, glycerin, sorbitol, mineral oil, isopropylmyristate, etc.,

buffer(s) (such as phosphate buffers, citrate buffers, and acetatebuffers, etc.) pH adjusters such as triethanolamine, potassiumhydroxide, sodium hydroxide), hydrochloric acid and phosphoric acidetc.,

gelling agents such as hydroxypropyl ethyl cellulose, hydroxyrthylcellulose, polyacrylic acid polymers, and poloxamers, etc.

vitamin(s) (e.g., A, B C, D, E, K, etc.),

mineral(s), plant extract(s) (e.g., aloe vera, witch hazel, elderflower,cucumber, chamomile, etc.),

anti-inflammatory agent(s),

emollient(s),

moisturizer(s),

skin protectant(s),

silicone(s),

analgesic(s),

skin penetration enhancer(s), such as propylene glycol, transcutol,isopropyl myristate,

colorant(s) such as yellow no. 5,

fragrance(s) (or perfume),

wax(es) (e.g., beeswax, paraffin wax, etc.),

propellant(s) (e.g., compressed air, hydrocarbons (such as propane,butane, isobutene, etc.),

sunscreen ingredient(s) (e.g., inorganic and/or organic sunscreens, suchas titanium oxides, zinc oxides, avobenzone, oxybenzone, homosalate,octocrylene octinoxate etc.), or

a combination comprising at least two of the forgoing.

For sunscreens, the formulation can contain 0.01 wt % to 20 wt %sunscreen ingredient(s), specifically, 0.1 wt % to about 10 wt %, andmore specifically, 0.5 wt % to 5 wt % based upon a total weight of theformulation. For skin products, those vehicles that are fat-soluble,i.e., those which can effectively penetrate skin layers and deliver theflavonoids to the lipid-rich layers of the skin. A formulationcontaining dispersed and/or solubilized flavonoids in an admixturecolloidal form can be added to a vehicle together with the oxides oftitanium and zinc such that the flavonoids will preferentially beabsorbed within the user's skin while the phase containing the zinc andtitanium oxides will not be absorbed but will form a protective UV filmbarrier external to the surface of the skin.

The concentrate of the invention can be loaded into a formulation byadding it into an oil/water (“o/w”) and/or water/oil/water (“w/o/w”)emulsion, which can comprise dispersant(s), emulsifiers, surfactants,and the like.

It is noted that, while the carrier can comprise a relatively simplesolvent or dispersant (such as oils and organic alcohols), it isgenerally preferred that the carrier comprise a composition moreconducive to topical application, and particularly one which will form afilm or layer on the skin to which it is applied so as to localize theapplication and provide some resistance to perspiration and/or one whichaids in delivery to the skin (e.g., to the skin's subsurface layers) andpenetration of the active ingredients into the lipid layers of the skin.Many such compositions take the form of lotions, creams, sprays andgels. Typical compositions include lotions containing water and/oralcohols, emollients (such as hydrocarbon oils, hydrocarbon waxes,silicone oils, vegetable fats and/or oils, animal fats and/or oils,marine fats and/or oils, glyceride derivatives, fatty acids, fatty acidesters, alcohols (e.g., polyhydric alcohols, alcohol ethers), lanolin(including derivatives), esters (e.g., polyhydric esters, wax esters),sterols, phospholipids, as well as combinations comprising at least oneof the foregoing), and generally also emulsifiers (nonionic, cationic oranionic). These same general ingredients can be formulated into a creamrather than a lotion, or into gels, by utilization of differentproportions of the ingredients and/or by inclusion of thickening agentssuch as gums or other forms of hydrophilic colloids.

In one embodiment, the formulation comprises the planar compound in boththe dissolved and dispersed (e.g., microparticulate) forms. Thedissolved form(s) can penetrate the skin layers to become bioactivewhile the dispersed compounds can serve as a reservoir for maintaining adissolved concentration level as the dissolved compounds are consumed soas to maintain sustained delivery.

A formulation can be prepared using a lecithin-based oil-in-water creamwith about 2.0 wt % apigenin and about 0.5 wt % ascorbic acid, withabout 0.5 wt % tocotrienol acetate and about 0.25 wt % glycolic acidwith the balance comprising the vehicle's components, based upon a totalweight of the formulation.

In another example, the formulation can be prepared using alecithin-based oil in water cream, 3.0 wt % with lecithin, about 0.5 wt% ascorbic acid, about 0.5 wt % tocotrienol acetate, about 0.25 wt %glycolic acid, and about a total of 8 wt % of the oxides of zinc andtitanium, with the balance comprising the vehicle's components, basedupon a total weight of the formulation.

Optionally, the composition can further comprise: (i) an additiveselected from the group consisting of surfactants, vitamins, minerals,plant extracts, anti-inflammatory agents, concentrates of plantextracts, emollients, moisturizers, skin protectants, humectants,silicones, skin soothing ingredients, skin penetration enhancers,colorants, perfumes (fragrances), preservatives, pH adjusters, and acombination comprising at least one of the forgoing; and/or (ii)titanium oxide, zinc oxide, or a combination comprising at least one ofthe forgoing.

Generally, the planar compound compositions can comprise greater than orequal to 0.01 weight percent (wt %) planar compound, specifically,greater than or equal to 1 wt %, for example, 0.1 wt % to 10 or 20 wt %,specifically, 0.5 wt % to 8 wt %, more specifically, 2 wt % to 5 wt %,based upon a total weight of the composition. The formulation cancomprise greater than or equal to 0.01 wt % (e.g., 0.01 wt % to 20 wt %planar compound, specifically, 0.05 wt % to 15 wt %, more specifically,0.1 wt % to 10 wt % planar compound, yet more specifically 0.5 wt % to 4wt % planar compound, and even more specifically, 1 wt % to 2 wt % basedupon a total weight of the formulation.

Ranges disclosed herein are inclusive and combinable (e.g., ranges of“up to 25 wt. %, or, more specifically, 0.5 wt. % to 5 wt. %”, isinclusive of the endpoints and all intermediate values of the ranges of“5 wt. % to 25 wt. %,” etc.). “Combination” is inclusive of blends,mixtures, alloys, reaction products, and the like. Furthermore, theterms “first,” “second,” and the like, herein do not denote any order,quantity, or importance, but rather are used to distinguish one elementfrom another, and the terms “a” and “an” herein do not denote alimitation of quantity, but rather denote the presence of at least oneof the referenced item. The suffix “(s)” as used herein is intended toinclude both the singular and the plural of the term that it modifies,thereby including one or more of that term (e.g., the film(s) includesone or more films). Reference throughout the specification to “oneembodiment”, “another embodiment”, “an embodiment”, and so forth, meansthat a particular element (e.g., feature, structure, and/orcharacteristic) described in connection with the embodiment is includedin at least one embodiment described herein, and may or may not bepresent in other embodiments. In addition, it is to be understood thatthe described elements may be combined in any suitable manner in thevarious embodiments. As used herein, the term “(meth)acrylate”encompasses both acrylate and methacrylate groups.

Examples of formulations of the invention are set forth in Table VI.

TABLE VI EXAMPLES OF FORMULATIONS CONTAINING THE DISCLOSED SOLUBILIZEDACTIVE AGENTS EXAMPLES OF FORMULATIONS FORMULATION CONTAINING THEDISCLOSED TYPES SOLUBILIZED ACTIVE AGENTS CAPSULES Capsules containingthe poorly soluble active agents & other active ingredients may includethe following ingredients: 0.01-10% of the disclosed active agents; and90-99.9% of inactive ingredients including oils, emulsifiers, solvents,saline solutions, preservatives Emulsions An emulsion is athermodynamically unstable (LOTIONS, system consisting of at least twoimmiscible liquid CREAMS), phases, one of which is dispersed in theother liquid & GELs phase. The system is stabilized by the presence ofan emulsifying agent. When the oil phase is dispersed throughout anaqueous continuous phase, the system is referred to as an oil-in-water(o/w) emulsion. When the oil phase serves as the continuous phase, theemulsion is referred to as water-in-oil (w/o) emulsion. Both lotions andcreams are emulsions. Creams are thicker than lotions. Gels consist of asolid three-dimensional work of a gelling agent that spans the volume ofa liquid medium. The key components include: 0.01-10% of the disclosedactive agents and other active ingredients; and 90-99.9% of otheringredients such as emulsifiers (surfactants), humectants, emollients,oils, fatty acids, solvents, stabilizing agents, gelling agents,preservatives, vitamins, penetration enhancers, dyes, fragrances, etc,are commonly added ingredients OINTMENTS Ointments are homogeneous,viscous semi-solid preparations. They are formulated using hydrophobic,hydrophilic, or water-emulsifying bases to provide preparations that areimmiscible, miscible, or emulsifiable with skin secretions. The keycomponents include: 0.01-10% of the disclosed active agents & otheractive ingredients; and 90-99.9% including the ointment base consistingof paraffins, beeswax, vegetable oils, fatty acids, stabilizers,emulsifiers, humectants, preservatives, fragrances, etc. DERMAL & Dermal& Transdermal Patches include the TRANSDERMAL following key ingredientscontained within a porous PATCHES matrix support: 0.01-10% of thedisclosed active agents & other active ingedients; and 90-99.9%including penetrating agents, preservatives, stabilizers, gellingagents, solvents such as short chain alcohols, pH adjusters, salinesolutions, etc. INJECTABLES Injectables include the following keycomponents: 0.01-10% of the disclosed active agents & other activeingredients; and 90-99.9% including preservatives, stabilizers, solventssuch as water and short chain alcohols, buffer, pH adjusters, salinesolutioms, etc. NASAL Nasal sprays may be atomized into a fine aerosolFormulations mist to include the following ingredients: (Solutions,0.01-10% of the disclosed active agents & other Sprays, gels active,ingredients; and and ointments) 90-99.9% including vegetable derivedoils, saline solutions, solvents, stabilizers, surfactants, buffers,preservatives, pH adjusters, gelling agents and petroleum etc.

Advantageous Topical Compositions

The following compositions are advantageous embodiments of theinvention. In other embodiments, another flavonoid, e.g. luteolin, orcombination of flavonoids, can be substituted for apigenin, and anothersurfacant can be substituted for polyorbate 80 to make the concentrate.In still further embodiments of the invention, other planar compounds ofthe invention, e.g. polyphenols, can be substituted for apigenin.

An apigenin formulation using a dimethyl sulfoxide-water solutioncomprising 1 to 20 wt % (advantageously about 10 wt %)apigenin/polysorbate-80 concentrate, 0 to 99.9 wt % (advantageouslyabout 45 wt %) dimethyl sulfoxide, and the balance comprising water,based upon a total weight of the formulation.

An apigenin gel formulation using a dimethyl sulfoxide-water solutioncomprising 1 to 20 wt % (advantageously about 10 wt %)apigenin/polysorbate-80 concentrate, 0 to 60 wt % (advantageously about45 wt %) dimethyl sulfoxide, 2 to 4 wt % (advantageously about 3 wt %)hydroxypropyl cellulose (quantity sufficient to achieve desiredviscosity), and the balance comprising water, based upon a total weightof the formulation.

An apigenin solution formulation using an ethanol-water solutioncomprising 1 to 20 wt % (advantageously about 10 wt %)apigenin/polysorbate-80 concentrate, 0 to 80 wt % (advantageously about66 wt %) ethanol, 0 to 20 wt % (advantageously about 10 wt % propyleneglycol), and the balance comprising water, based upon a total weight ofthe formulation.

An apigenin gel formulation using an ethanol-water solution comprising 1to 20 wt % (advantageously about 10 wt %) apigenin/polysorbate-80concentrate, 0 to 80 wt % (advantageously about 66 wt %) ethanol, 0 to20 wt % (advantageously about 10 wt %) propylene glycol, gelled witheither ˜0.5 to ˜2.5 wt % hydroxyethyl cellulose or ˜0.5 to ˜2.5 wt %sodium hyaluronate or ˜0.5 to ˜2 wt % carbopol (quantity sufficient toachieve desired viscosity), and the balance comprising water, based upona total weight of the formulation.

An apigenin emulsion formulation comprising 1 to 20 wt % (advantageouslyabout 5 to 10 wt %) apigenin/polysorbate-80 concentrate, 0 to 20 wt %(advantageously about 10 wt %) ethoxydiglycol, 0 to 20 wt %(advantageously about 12 wt %) myristyl lactate, 0.1 to 1.0 wt %(advantageously about 0.4 wt %) carbopol 980, 0.1 to 1.0 wt %(preferably 0.3 wt %) Pemulen TR1, ˜0.17 wt % methylparaben, ˜0.03 wt %propylparaben, ˜0.1 wt % EDTA, 0 to 15 wt % (advantageously about 10 wt%) cyclomethicone, 0 to 10 wt % (advantageously about 2 wt %) oleylalcohol, ˜0.1 wt % butylated hydroxytoluene, and the balance comprisingwater (with the final pH adjusted to ˜6.5 using dilute HCl or a 10%sodium hydroxide solution), based upon a total weight of theformulation. Pemulen polymers are high molecular weight, crosslinkedcopolymers of acrylic acid and C10-C30 alkyl acrylate. Carbopol 980 is acrosslinked polyacrylate polymer.

Nutraceuticals/Dietary Supplements

A solubilized compound, such as a polyphenol concentrate, or formulationof such concentrate, can be used for many nutraceutical products such asisolated nutrients, fortified foods and dietary supplements. As usedherein, a nutraceutical is any nontoxic food extract supplement that hasscientifically proven health benefits for both the treatment andprevention of disease.

Food/Medical Foods

Medical foods are formulated to be consumed or administered internallyunder the supervision of a physician. They are intended for the specificdietary management of a disease or condition for which distinctivenutritional requirements, on the basis of recognized scientificprinciples, are established by medical evaluation. Medical foods can beingested through the mouth or through tube feeding. Medical foods arealways designed to meet certain nutritional requirements for peoplediagnosed with specific illnesses. A planar compound concentrate, suchas flavonoid concentrate, or a formulation thereof, can be used inmedical foods. For example, the addition of a polyphenol (e.g.flavonoid) concentrate to beverages (e.g. alcohol types) will serve as ameans for the oral delivery of poorly soluble but beneficialpolyphenols. Also included in the invention are functional beverages andfunctional shots, genetically engineered “designer” food, herbalproducts, and processed products such as cereals, soups, and beverageswhich include a concentrate.

Cosmetics/Cosmeceuticals

The concentrates or formulations of the subject invention, e.g.including polyphenol concentrates, can be used in many products such ascosmetic and dermatological products, including foundations, sunscreenproducts, sunless skin tanning products, hair removal products, creams(e.g., moisturizing creams, burn creams, skin benefit creams, nightcreams, dermatological creams, etc.), serums, liquids, skin benefitlotions, softeners, gels, sprays, foams, soltions, soaps, shampoos,ointments, lipsticks, cleansers, toners, masks, hair products, fingernail products, as well as other cosmetic products or applications.

IV— Kits

According to another aspect of this invention, kits for forming aformulation for delivery of a bioactive agent are provided. The kits ofthe invention comprise a first aliquot portion of a concentratesolution, and a second aliquot portion of a carrier. When mixed, thefirst and second aliquot portions form a composition for delivery of thebioactive agent contained therein. A preservative may be incorporatedinto one of the first and second aliquot portions. The carrier istypically a dermatological, oral, injectable, or aerosol carrier.

The kits of the invention can advantageously be provided with a doublesyringe having first and second syringe barrels which respectivelycontain the first and second aliquot portions of the reaction mixture. Aduel syringe set-up can be used in preparation of the formulationsaccording to the present invention. For example a concentrate solutionof a flavonoid, such as apigenin, can be contained within the barrel ofone syringe, while a carrier can be contained within the barrel of theother syringe. The syringes are connected to one another so that thecontents can be mixed by alternately transferring the mixture from onesyringe barrel to another for about 30 cycles. The concentrate solutionis stable for at least one year and the carrier stable for at least thattime, in some cases indefinitely. Maintenance of sterility, broadapplicability, stability of the bioactive agent and ease of use are theattributes of such a double syringe system.

V—Uses of the Compounds and Formulations of the Invention

Flavonoids

Flavonoids have multiple therapeutic applications since they are freeradical scavengers, anti-oxidants, superoxide anions, UV absorbers,vasodialators, anti-hyaluronidase (inhibits breakdown of hyaluronicacids by inhibiting hyaluronidases), anti-collagenase (inhibitsbreakdown of collagen by inhibiting collagenases), anti-elastase(inhibits breakdown of elastin by inhibiting elastases), and scavaginglipid peroxide radicals. Flavonoid compounds are also known to beeffective in strengthening collagen structures. Further, flavonoids haveanti-mutagenic, anti-angiogenic, anti-carcinogenic, anti-inflammatory,antibacterial and antiviral effects. The anti inflammatory effectsinclude inhibition of TNF-alpha, IL-beta, COX-2, protein kinase PKC,iNOS, and T helper cells Th 1 and Th 17. Flavonoids, apigenin inparticular, is a stimulator of p53. Researchers have found that apigenininduces reversible, cell-cycle arrests at G1 and G2/M phase of the cellcycle.

Flavonoids alone or in combination with other preventive and/ortherapeutic effective drugs, are effective in treating or preventing inmammals, including humans, the most common diseases such as cancer,autoimmune disease, diabetes, ulcer, cardiovascular disease,atherosclerosis, and liver disease. The compounds also haveantithrombogenic activity.

Skin Diseases

This disclosure provides methods for making topical formulationscontaining flavonoids, such as apigenin, at a pharmaceuticallymeaningful concentration in a dermatologically acceptable pH range. Theflavonoids are in dissolved form. The topical application formulationcan be a composition in the form of a gel, ointment, solution, lotion,cream, spray, dermal patch, transdermal patch and so forth, so as todeliver sufficient flavonoid into mammalian (such as human) tissue(e.g., into mammalian keratinous tissue).

Topical Application Amount

A typical topical dose ranges from 1 to 10 mg/cm², preferably 1 to 5mg/cm² and most preferably from 1 to 3 mg/cm². The dosage variesaccording to condition and mode of administration The topical dose usedin FDA sunscreen topical testing is 2 mg/cm² of formulation applied toexposed skin. “Re: Tentative Final Monographfor OTC Sunscreen”, Food andDrug Administration (U.S.). 1998-09-11. Retrieved 2009-09-25 Providedone assumes an “average” adult build of height 5 ft 4 in (163 cm) andweight 150 lb (68 kg) with a 32 in (82 cm) waist, that adult wearing abathing suit covering the groin area should apply 29 g (approximately 1oz) evenly to the uncovered body area. Considering only the face, thistranslates to about ¼ to ⅓ of a teaspoon for the average adult face.Larger individuals should scale these quantities accordingly.

In terms of the amount of topical medication that generally should beapplied to affected skin, dermatologists refer to the “fingertip unit”as the recommended guidance. One fingertip unit is the amount of topicalformulation that is squeezed out from a standard tube along an adult'sfingertip. One fingertip unit is approximately 500 mg of formulation(tube with a standard 5 mm nozzle), and recommendations for the numberof units needed to cover affected areas are offered. For example, threefingertip units are required to adequately cover psoriasis on the scalp,whereas eight fingertip units are needed for the entire leg and foot.This method provides a means for patients to more accurately dose theirtopical medications.

Prevention and Treatment of Skin Damage Due to Solar Radiation

Soluble forms of the flavonoid, e.g. apigenin and/or luteolin, canreadily penetrated into and be absorbed by the skin to prevent damage(photoaging) or to repair the skin matrix that has been damaged. Asshown in Example 15 below, the formulations of the subject inventionallow significant skin penetration of the flavonoid.

The low solubility of apigenin and/or luteolin in the excipientstypically comprising lotions and creams makes formation of suchcompositions with desired amounts of flavonoid in solution difficult. Inone embodiment, the formulation contains a sufficient amount ofsolubilized flavonoid at a nearly neutral pH to penetrate into theliving skin matrix to minimize or eliminate skin tissue damage toprotect living skin from damage caused by exposure to UV rays and/orpre-penetrate. The topical formulations can be administered to anindividual, preferably by topical application to the skin of theindividual, orally (e.g., as a food supplement), etc. The formulationscan be administered in an amount effective to prevent UV damage, e.g.,to inhibit free radicals, reactive oxygen species, and/or otheroxidizing species.

With respect to its anti skin cancer activities, apigenin actseffectively even in very low concentrations, <about 50 μM. Apigeninexhibits antiproliferative and cytotoxic effects by affecting apoptosisand necrosis mechanisms during cell proliferation and angiogenesis thatare the major characteristics of a variety of cancer cells includingprostrate cancer, breast cancer, lung cancer, leukemia, thyroid cancerand liver cancer, resulting in the inhibition of proliferation of cancercells.

Mechanism

The primary mechanisms of flavonoids, e.g. apigenin, are believed to betheir capability to increase the stability of p53; thereby inducing bothG1 and G2/M cell cycle arrests and its well documentedanti-inflammatory, anti-oxidant, nontoxic, and non-mutagenic properties.These cell cycle arrests are fully reversible after removal of apigeninby washing or its diffusion out of the skin.

In light of the fact that apigenin causes both G1 and G2/M cell cyclearrests, the essence of apigenin's chemopreventative activity may be toinhibit cancer initiation and progression by ensuring that sufficientintrinsic and artificially imposed cell cycle checkpoints exist in thepresence of DNA damaging and tumor promoting agents. Apigenin andluteolin treatment of skin prior to sunlight exposure may extend thetime cells normally arrest in G1 and G2/M in response to DNA damage.These flavones increase the duration of the G1 phase beyond that whichoccurs in normal cells in response to DNA damage, or alternatively,these flavones retard cells containing an activated oncogene in G1 whenotherwise cell cycle progression would continue even in the presence ofsubstantial DNA damage. Hence, the time spent in G1 and G2/M is criticalfor cells to efficiently repair all DNA mutations, and thus slow orprevent the carcinogenic process.

Since the effects of sunlight damage are cumulative over a lifetime, thetumor suppressor protein p53, which is the most commonly mutated gene inall human and animal cancers, may already be inactivated in somekeratinocytes by the time a person uses a topical application ofapigenin and/or luteolin. Since the effects of these flavones arep53-dependent on the G1 arrest and p53-independent on G2/M arrest, ininstances where keratinocytes already have an inactivated p53 gene,apigenin will bolster the G2/M arrest in these small foci ofpremalignant cells in order to prevent additional mutations,translocations, and/or chromosome loss during mitosis. In addition,apigenin and/or luteolin may exert its protective effects by scavengingfree radicals generated in response to UV-B/A sunlight irradiation.

It is believed that apigenin treatment can enhance the apoptoticresponse initiated by UVB. Without being bound by theory, it is believedthat the chemo-preventive action of apigenin is explained by its abilityto enhance UV-induced apoptosis by significantly increasing thestability of p53 which is a prime factor in the skin cancer apoptosisprocess. Therefore, there is a need to deliver apigenin into the viableepidermis or the whole skin layer at a pharmaceutically meaningfulconcentration in order to be effective in skin cancer prevention. (LiB.; Birt D. F.; Pharmaceutical, Volume 13, Number 11, November 1996, pp.1710-1715(6))

As disclosed herein, a composition for the topical applicationcontaining flavonoids, particularly apigenin and/or luteolin, is usefulfor the prevention and/or treatment of skin damage ansing from exposureto solar radiation (UVA and/or UVB). Apigenin and/or luteolincompositions also augment the efficacy of other ingredients in topicalcompositions for sunburn prevention and treatment.

In use, the product can be used in single or multiple applications toattain the desired results. In some embodiments, the sunscreeningredients can be part of the formulation, and/or can be applied as asecondary application such that a film containing the sunscreeningredients serves to provide additional full spectrum UV radiationprotection by blocking or reflecting UV radiation.

Since apigenin and luteolin function intracellularly on the cell cycle,either could be combined with other sunscreen agents that functionsimply as a barrier on the outside of the cell to absorb, block orreflect UV energy in sunlight. Thus, topical application of apigeninand/or luteolin, reversible cell cycle regulators, represents a usefuland novel approach for skin cancer prevention and can be usedsequentially or in combination with currently marketed topical sunscreenproducts.

These flavonoids are exceptionally beneficial as additives to topicalformulations for their anti-oxidant, anti-inflammatory, UV skinprotection and other desirable properties. Thus, topical application ofapigenin and luteolin represent a useful and novel approach for skincancer prevention/treatment and could be used prior to or in combinationwith currently marketed topical sunscreen products.

Not to be limited by theory, it is believed that the formulations can beemployed, for example, to treat or prevent skin cancers caused byexposure to ultraviolet (UV) light or sunlight.

Disclosed herein are compositions containing flavonoids or topicalapplications for the prevention and/or treatment of skin cancer andother topical cancers including but not limited to cervical and breastcancer. The composition contains pharmaceutically sufficient amount ofapigenin to penetrate into the skin layer, e.g., to increase thestability of p53 to prevent and treat skin and other topical cancers.

It is believed that the UVB photo-protective effects of the antioxidantapigenin and luteolin are significant when applied in distinct mixturesin appropriate vehicles. Flavonoid(s) together with other ingredientsprovide a natural approach to efficiently supporting the body's owndefense mechanism in providing protection from sunburn and chronic UVdamage. The natural antioxidant properties and anticancer properties ofapigenin and/or luteolin combined with mineral pigments provide asynergistic, photo-protective effect to reduce the risk of UV damage andskin cancer. The other natural ingredients including antioxidants suchas vitamin E and moisturizes can be added to create a synergy thatenhances UV protection and also soothes the skin.

Also disclosed herein are methods of reducing and/or preventing theeffects of sun exposure which can comprise: applying a topicalformulation comprising a flavonoid and a dermatologically acceptablecarrier to permit delivery of the flavonoid components to mammaliankeratinous tissue. Optionally, the topical cosmetic composition can beapplied a second time, a third time, or more.

Cancer

Three ideal qualities of a cancer chemopreventative agent are: 1) thatit is a natural compound present in foods known to be associated withreduced cancer incidence; 2) that it has a known mechanism of action;and 3) that the effects are reversible. It is believed that flavonoidssuch as apigenin and luteolin satisfy all three criteria.

The formulations of the invention can be used for cancer prevention aswell as cancer treatment. The formulations are useful for the treatmentor prevention of skin cancers (including actinic keratosis, melanoma,basal cell carcinoma), ovarian cancer, cervical cancer, prostate cancer,breast cancer, lung cancer, leukemia, thyroid cancer, liver cancer andbrain cancer including neuroblastoma.

Methods of Treatment of Other Skin Disorders

The compounds and formulations of the invention are useful for thetreatment of psoriasis. Example 15 demonstrates that topicalformulations penetrate human skin in a concentration sufficient to be oftherapeutic value.

Additional dermatological disorders and related afflictions/conditionsthat can be treated or prevented by the topical use of the formulationsand compositions of this invention include, but are not limited to thefollowing: acne, alopecia, atopic dermatitis/eczema, cutaneous lupuserythematosus, dermal sensitization and irritation, dry skin (xerosis,ichthyosis), fungal infections, and rosacea, contact dermatosis,autoimmune afflictions including psoriasis, and arthritis. The topicaladministration of apigenin/flavonoids allows excellent bioavailability.Hence, these topical formulations are alternatives to costly and lessdesirable steroids and cytotoxic drugs.

Methods of Treatment of Other Disorders

The compounds, compositions and formulations of the invention can alsobe used for the treatment of other autoimmune disease such as lupus,arthritis, allergies and asthma. Flavonoid formulations of the inventionrepresent new adjuvant therapeutics with efficacy in autoimmune disease.The bioavailability of dietary plant-derived COX-2 and NF-kB inhibitors,such as apigenin is valuable for suppressing inflammation in lupus andother Th17-mediated diseases like rheumatoid arthritis, Crohn's disease,and psoriasis, and in prevention of inflammation-based tumorsoverexpressing COX-2 (e.g. colon, breast). Apigenin suppresses lupus byinhibiting autoantigen presentation for expansion of autoreactive Th1and Th17 cells. The formulations of this invention offer a novel meansof delivering apigenin/flavonoids for the treatment of autoimmuneindications/diseases. The administration can be as an adjunct to otherautoimmune therapies such as an anti-TNF antibody (e.g. for psoriasis orfor rheumatoid arthritis).

The compounds and formulations are also useful for the treatment ofneurological and neurodegenerative disorders. Several research studieshave provided support for apigenin and luteolin's anti-inflammatoryeffects and their neuroprotective/disease-modifying properties invarious neurodegenerative disorders, including Alzheimer's disease.

In another embodiment, the compounds and compositions of this inventionare useful for the treatment of allergic diseases as well as bacterialinfections.

Examples of the TNFα related conditions that can be treated, preventedor ameliorated with the flavonoids of the invention include, but are notlimited to, rheumatoid arthritis, juvenile rheumatoid arthritis,osteoarthritis, spondyloarthropaties, inflammatory bowel disease,chronic heart failure, diabetes mellitus, systemic lupus erythematosus,scleroderma, sarcoidosis, polymyositis/dermatomyositis, psoriasis,multiple myeloma, myelodysplastic syndrome, acute myelogenous leukemia,Parkinson's disease, AIDS dementia complex, Alzheimer's disease,depression, sepsis, pyoderma gangrenosum, hematosepsis, septic shock,Behcet's syndrome, graft-versus-host disease, uveitus, Wegener'sgranulomatosis, Sjogren's syndrome, chronic obstructive pulmonarydisease, asthma, acute pancreatitis, periodontal disease, cachexia,cancer, central nervous system injury, viral respiratory disease, andobesity.

Examples of the IL-1β related conditions to be treated, prevented orameliorated with the flavonoids of the invention include, but are notlimited to, rheumatoid arthritis, hematosepsis, periodontal disease,chronic heart failure, polymyositis/dermatomyositis acute pancreatitis,chronic obstructive pulmonary disease, Alzheimer's disease,osteoarthritis, bacterial infections, multiple myeloma, myelodysplasticsyndrome, uveitis, central nervous system injury, viral respiratorydisease, asthma, depression, and scleroderma.

Due to the inhibitory activity of flavonoids on IL-4 and IL-13synthesis, it can be expected that the intake of flavonoids, dependingon the quantity and quality, can ameliorate allergic symptoms or preventthe onset of allergic diseases. (Int Arch Allergy Immunol. 2004 June;134(2): 135-40.)

Apigenin possesses anti-inflammatory activity in human periodontalligament (hPDL) cells and works through a novel mechanism involving theaction of heme oxygenase-1 (HO-1) 1. Thus, apigenin has benefits as ahost modulatory agent in the prevention and treatment of periodontaldisease associated with smoking and dental plaque. (Gil-Saeng Jeong etal.; Anti-inflammatory effects of apigenin on nicotine-andlipopolysaccharide-stimulated human periodontal ligament cells via hemeoxygenase-l., International Immunopharmacology, Vol.: 9, November 2009).

In another embodiment, the compounds and formulations of this inventioncan be useful for promoting hair growth. Research studies teach that theapigenin stimulates hair growth through downregulation of the TGF-beta1gene.

The formulations of this invention are also useful in the treatment andprevention of arteriosclerosis.

Other Active Agents

Formulations made using the methods of the invention of the compoundsnote in Section I above “Compounds of the Invention” can be used for theindications (uses) specified or known for those compounds. For example,a formulation of camptothecin made using polysorbate as in the subjectinvention, can be used in the treatment of cancer.

EXAMPLES Example 1. Solubility in Polysorbates

The Apigenin and Polysorbate 80 resulting product is referred to as“A/PS80”. A/PS80 was formed as follows:

-   -   The unprocessed apigenin powder & viscous liquid polysorbate 80        (PS80) were mixed in the ratio from about 5 to 10 wt % of        apigenin to 95 to 90 wt % polysorbate 80 and a small quantity of        D.I. water and optionally acetone and/or ethyl alcohol in a        beaker.    -   This mixture was then thoroughly stirred to form a thick        paste-like blend.    -   The mixture was then slowly heated to relatively high        temperatures while stirring. The heating was accompanied by the        boiling off of the water and also volatile constituents present        in the polysorbate 80. The heating process was conducted with        care to avoid the mixture overflowing from the beaker due to        foaming resulting from the heating process.    -   Upon the removal of the volatiles and heating to temperatures in        excess of about 200 to 300° C., a dark brown transparent liquid        resulted such that all the solid apigenin is solubilized in the        polysorbate 80 mixture.    -   Upon cooling to ambient temperatures, a viscous brown clear        liquid resulted. The higher the apigenin content—the darker the        resulting color). Addition of a few crystals of apigenin to the        cooled A/PS80 liquid did not result in precipitation; thereby        demonstrating that the A/PS80 liquid is not supersaturated.        According to the published solubility results shown in the Table        VII the solubility of apigenin in water, ethyl alcohol and Span        80 are listed as follows: (Ref Li et al, J of Pharm Sci, Vol.        86, No. 6, June 1997).

TABLE VII Solubility of Apigenin @ 25° C. SOLUBILITY SOLVENT (mg/ml)(ppm) Water 0.00135 1.35 Ethyl Alcohol 1.65 1,630 Span 80 0.15 150

The concentration of apigenin in A/PS80 was measured by HPLC-MS. Basedmeasured the concentration of apigenin in A/PS80 on the calculated valueof 4.05% concentration of apigenin in the viscous A/PS80 liquid; thecontent of apigenin is 40.5 mg/ml or 40,500 ppm.

The following paragraphs list experimental observation attributable toA/PS80.

-   -   The addition of A/PS80 to the standard hydrated apigenin lotions        (which contain a substantial concentration of nanoparticulates)        contributed to an enhancement in saturation soluble        concentration levels. The enhanced solubility level was        qualitatively determined via colorimetric testing performed on        filtrate liquids passing through a 0.2 micron filter.    -   Addition of A/PS80 to Purell (the widely used bactericidal        fluid) resulted in an appreciable apigenin soluble level        attributable to the high ethyl alcohol content of Purell. The        soluble apigenin levels achieved with polysorbate 80 were        significantly greater than both hydrated apigenin and        unprocessed apigenin.    -   Experiments where A/PS80 was added to Purell followed by the        application of the apigenin lotion worked quite well. The idea        was to take advantage of ethyl alcohol's favorable penetrating        and solubility properties (note that ethyl alcohol will        evaporate shortly after application which will tend to dry out        the skin) followed by the application of our apigenin formation        to assist in skin re-hydration.

The concentrations of apigenin in weight % for selected solvents asdetermined by LCMS (Liquid Chromatography-Mass Spectroscopy) shown inTable VIII.

TABLE VIII APIGENIN ANALYTICAL SUMMARY IN SELECTED SOLVENTS SOLUBLEAPIGENIN CONCENTRATION SAMPLE DESCRIPTION (% Wt/Wt) - (mg/ml)Apigenin/PS80 added to Purell Lotion  0.52%-5.2 mg/ml Apigenin/PS80added to Ethyl Alcohol  0.91%-9.1 mg/ml Rubbing Solution Apigenin/PS804.05%-40.5 mg/ml (Concentrated Stock Solution)

Additional testing verified that essentially there was insignificantdecomposition products resulting as a consequence of heating PS80 withapigenin to elevated temperatures approaching 250-300° C.

FIG. 2 shows a statistical analysis of PS 80 prior to heating ascompared to the A/PS80 solution. Insignificant differences were observedbetween the control sample and the invention sample showing that thesurfactant had not degraded. In FIG. 2, each component detected isrepresented by a dot. PS80 is a polymer and as such, shows manyoligomers. This explains the large number of components or dots on theplot. The Xs are indicative of mass features which are distinct to theA/PS80 sample statistically. Very few distinct features were observedindicating that the PS80 did not significantly degrade. The presence ofan only a few Xs indicates few differences between the control andinvention samples.

Example 2. Solubility of Selected Flavonoid Concentrates in Polysorbate80 and PEG 400 as Determined by HPLC

The objective of this work was to quantify a total of six differentflavonoids in PS80 and PEG-400. The samples were also analyzedqualitatively to look for signs of degradation of the solvents due tothe elevated temperatures required of the thermal treatment process.

This project was divided into three distinct phases: methodvalidation/development, quantitation of the individual flavones, andqualitative analysis to look for signs of PS80/PEG400 degradation.Method development was performed to determine the suitability of apreviously used HPLC method for Apigenin for the remaining flavonoids tobe quantified in either PEG400 or PS80.

Table IX provides the HPLC measured flavonoid concentration in theas-prepared flavonoid concentrates formulated by the thermal treatmentprocess in both PS80 and PEG-400.

TABLE IX Flavonoid Analytical Summary In Selected Solvents Flavonoid inFlavonoid Solvent Solvent - (mg/ml) Quercetin PS80 65.0 Resveratrol PS80127.5 Rutin PS80 14.3 Luteolin PEG-400 72.5 Apigenin PEG-400 34.8Hesperidin PEG-400 81.0 ± 8.0

Statistical Analysis software was used to look for the presence ofunique features in the various flavonoid samples of Table IX whencompared to the control PS-80 and PEG-400 reference materials. LiquidChromatography Mass Spectroscopy (LCMS) was used to determine PS80 andPEG-400 degradation products. The number of unique components in eachsample ranged from 12 in the Hesperidin-PEG-400 sample to 55 in theApigenin-PEG-400 sample. A small number of unique components wasexpected due to the addition of the flavonoid and any trace componentsintroduced with the flavonoid. Thus, the low number of unique componentsindicates that no appreciable degradation occurred.

Example 3. Additional Flavonoid Polysorbate Formulations

In addition to apigenin, testing with Polysorbate 80 was expanded toinclude several flavonoid compounds. Table X includes the chemical andphysical property data of the flavonoids selected for solubility testingwith Polysorbate 80.

TABLE X SUMMARY OF CHEMICAL & PHYSICAL PROPERTIES OF FLAVONOIDS TESTEDWATER MP SOLUBILITY PARTIAL LISTING OF COMPOUND MW (° C.) (mg/ml)FLAVONOID SOURCES APPEARANCE APIGENIN 270 ~360 * 0.00002 Parsley, Thyme,Celery, Yellow Crystalline (>Sol. In alcohol) Chamomile Powder LUTEOLIN286 ~330 * 0.38 mg/ml Celery, Oregano, Thyme, Yellow Powder (>Sol. Inalcohol) Chamomile RESVERATROL 228 ~255 * 0.1 to 0.3 mg/ml Red Grapes &Red Wine, White Powder with 50 mg/ml in alcohol Peanuts, Some Berries aslight yellow cast QUERCETIN 302 ~315 * <1 mg/ml Apples, Tea, Citrus,Yellow Crystalline Broccoli, Berries Powder HESPERIDIN 610 ~260 * Valuescited from Buckwheat, Citrus, White to Yellow 0.05 to 3 mg/ml Cherries,Grapes Powder RUTIN 610 ~242 * 0.07 mg/ml Buckwheat, Citrus, Berries,Yellow to Green Tea Powder * saturation concentrations solubility varieddepending on published sources

Table XI contains a summary of the Polysorbate 80 solubility testingresults with a variety of flavonoids.

TABLE XI SUMMARY OF FLAVONOID TESTING WITH PS80 SOLUBLE CONC. RANGE (%wt/wt)/ COMPOUND PS80 SOLUBILITY COMMENTS (mg/ml) APIGENIN 1. ThermalTreatment method. 1. 4-6%/ (Noted in Example 1) (40-60 mg/ml)LUTEOLIN 1. Thermal Treatment method. 1. >8% (>80 mg/ml) The upper sol.limit was not determined. 2. Luteolin in H2O & PS80 slurry 2. Up to ~5%was boiled resulting in solubilizing (~50 mg/ml) luteolin but to <<extent than the Thermal Treatment Method RESVERATROL 1. ThermalTreatment method 1. >8% (>80 mg/ml) 2. The H2O boiling method utilized2. Dissolution for Luteolin did not dissolve not detectable Resveratrol.QUERCETIN 1. Thermal Treatment method 1. >7% (>70 mg/ml) The upper sol.limit was not determined. 2. Quercetin in H2O & PS80 slurry 2. <<7% wasboiled resulting in solubilizing quercetin but to << extent than theThermal Treatment method. HESPERIDEN 1. The Thermal Treatment Method 1.~<2% resulted in a slight amount of a gray (~<20 g/ml) coloredprecipitate which was Decomposition removed by filtration with a 0.2micron filter. 2. A 5% Hesperidin to PS80 ratio 2. ~<1% added to water.The mixture was (~<10 mg/ml) boiled to form solubilized Hesperidin. Noevidence of the “enhanced aqueous solubility” was noted. RUTIN 1. TheThermal Treatment Method 1. ~<1% resulted in a slight amount of a gray(~<10 mg/ml) colored precipitate which was removed by filtration with a0.2 micron filter. 2. A 5% Rutin to PS80 ratio added 2. ~<0.5% to water.The mixture was boiled to (~<15 mg/ml) form solubilized Rutin. Noevidence of the “enhanced aqueous solubility” was noted.

Example 4. Solubility in Polysorbates Other than Polysorbate 80 Via theElevated Temperature Processing Method

Non-ionic surfactants are extensively used in cosmetics and foodsbecause they are considered to be harmless because they are fatty acidesters of polyalcohol such as sorbitan, sucrose, and glycerin.Consequently, it was decided to evaluate a number of suitable nonionicpolysorbate structured surfactants to enhance the saturation solubilityconcentration via the high temperature processing methods disclosed inExample 1.

Table XII lists several nonionic surfactants consisting of PEG-ylatedsorbitan (a derivative of sorbitol) esterified with fatty acids. Allsurfactants tested were oily liquids which satisfied the criteria ofremaining stable at temperatures >200° C. Similarly, all testedflavonoids including apigenin were selected on the basis of having poorsolubility properties.

It was observed that the flavonoid slurry mixture changes in bothparticulate solubility and color (a dark brown-red) was observed whentemperature levels exceeded 200 to 300° C. Addition of a few crystals ofthe apigenin to the cooled liquid did not result in precipitation;thereby demonstrating that the liquid is not supersaturated.

The nonionic surfactants listed in Table XII are arranged in order ofascending (Hydrophile-Lipophile Balance) HLB values. HLB is an empiricalexpression for the relationship of the hydrophilic (“water-loving”) andhydrophobic (“water-hating”) groups of a surfactant. The higher the HLBvalue, the more water-soluble is the surfactant. The majority arelotions (oil-in-water emulsions) or creams (water-in-oil emulsions). Themost common emulsion type, oil-in-water (o/w), often requires higher HLBsurfactants—preferably 12-16 while water-in-oil emulsions (w/o) requirelow HLB surfactants—preferable 7-11. Surfactants with an HLB value <10are oil soluble while those >10 are soluble.

As noted in Table XII, Span 20 is very suitable for water-in-oil topicalformulations while Polysorbate 80 would be most appropriate forsolubilizing apigenin in oil-in-water topical formulations.

TABLE XII A Summary of Apigenin Solubility in Nonionic Surfactants viathe High Temperature Processing Method High Apigenin Temp. LiteratureApigenin Solubility NONIONIC CHEMICAL Solubility @ (° C.) - HLBSURFACTANTS NAME (mg/ml) (mg/ml) VALUE USES Span 80 Sorbitan ~8 0.15 4.3Foods, beverages, monostearate Pharmaceuticals Span 20 Sorbitan ~10 0.178.6 Foods, beverages, monolaurate Pharmaceuticals Nonoxynol-9Polyoxyethylene ~30 — 13.0 Disinfection, nonyl phenyl spermicide, ethercosmetics Polysorbate 60 Polyoxyethylene ~15 — 14.9 Foods, beverages,(20) sorbitan Pharmaceuticals monostearate Polysorbate 80Polyoxyethylene ~50 — 15.0 Foods, beverages, (20) sorbitanPharmaceuticals oleate Polysorbate 20 Polyoxyethylene ~25 — 16. 7 Foods,beverages, (20) sorbitan Pharmaceuticals monolaurate *Propylene — ppt.1.0 — Foods, beverages, Glycol est. ~1 pharmaceuticals Note: *Not asurfactant

Example 5. The Solubility of Active Agents in PS80 and PEG 400 Via theThermal Treatment Method

A study was undertaken to investigate the potential aqueous solubilityenhancement of active agents dissolved in PS80 and PEG 400 by means ofthe disclosed thermal treatment process.

Table XIII briefly summarizes solubility results of 4 active agentsemploying the disclosed thermal treatment process to form concentratesthat resulted in the enhancement of aqueous solubility of severalrelatively insoluble flavonoids.

TABLE XIII A Summary of the Active Agent/Surfactant Testing Utilizingthe Thermal Treatment Process ACTIVE AGENT MP PS80 Solubility—Results/PEG400 Solubility—Results/ COMPOUND USAGE MW (° C.) Comments Comments

Pain Relief/ NSAID 241 230 1 wt % solubilized in PS80 @ ~140° C. &remained solubilized @ Room Temp. ⁽¹⁾Not Sup.Sat. Most Mefenamic Aciddissolved when H₂O was added to 1 wt. % solution. 1 wt % solubilized inPEG400 @ ~140° C. & remained solubilized @ Room Temp. ⁽¹⁾Not Sup.Sat.Most Mefenamic Acid ppt. out when H₂O was added to 1 wt. % solution.

Pain Relief/ NSAID 318 283 5 wt % solubilized in PS80 @ ~200° C. &remained solubilized @ Room Temp. ⁽¹⁾Not Sup.Sat. Diclofenac Sodiumremained clear when H₂O was added to the 10 wt % Sol.—(pH ~7). Addingcitric acid sol. to a pH ~4 pptd. diclofenac sodium. 5 wt % solubilizedin PEG400 @ ~100° C. & remained solubilized @ Room Temp. ⁽¹⁾Not Sup.Sat.Diclofenac Sodium remained clear when H₂O was added to the 10 wt %Sol.—(pH ~7). Adding citric acid sol. to a pH ~4 pptd. diclofenacsodium.

Pain Relief/ NSAID 296 177 4 wt % solubilized in PS80 @ ~140° C. &remained solubilized @ Room Temp. ⁽¹⁾Not Sup.Sat. Diclofenac Acidremained clear when H₂O was added to the 10 wt % Sol.—(pH ~7.5). A clearsol. remained after citric acid sol. added to a pH ~4 4 wt % solubilizedin PEG400 @ ~140° C. & remained solubilized @ Room Temp. ⁽¹⁾Not Sup.Sat.Diclofenac Acid pptd. when H₂O was added to the 4 wt % Sol.— (pH ~7.5).

Anti Cancer/ Keratosis 130 282 10 wt % solubilized in PS80 @ ~200° C. &remained solubilized @ Room Temp. ⁽¹⁾Not Sup.Sat. Fluorouracil remainedclear when H₂O was added to the 5 wt % Sol.—(pH ~7). A clear sol.remained after citric acid sol. added to a pH ~4 10 wt % solubilized inPEG400 @ ~175° C. & remained solubilized @ Room Temp. ⁽¹⁾Not Sup.Sat.Fluorouracil remained clear when H₂O was added to the 5 wt % Sol.—(pH~7). A clear sol. remained after citric acid sol. added to a pH ~4 Note:⁽¹⁾The solution was not supersaturated as determined by the addition ofa crystal such the precipitation did not occur.

The results of this study indicated that the thermal treatment processis useful to improve solubility, dissolution rate, and subsequently,bioavailability of poorly soluble drugs.

Example 6. Solubility in Ceteareth-20 Via the Elevated TemperatureProcessing Method

Solubility testing with Ceteareth-20, a nonionic surfactant, and theflavonoids, apigenin, luteolin and quercetin, were subjected to thethermal treatment process to investigate the likelihood of solubilityenhancements in aqueous solutions.

Ceteareth-20 (CAS #68439-49-6) was obtained from Making Cosmetics.com,Inc located in Renton, Wash. Ceteareth-20 is a polyoxyethylene ether ofhigher saturated fatty alcohols (cetyl/stearyl alcohol). At roomtemperature, Ceteareth-20 is a solid, has no odor and melts at 40° C.Ceteareth-20 forms oil-in-water emulsions and has an HLB value rangingfrom 15-17.

For each of the flavonoid solubility tests, the following procedure wasfollowed:

-   -   1. Weigh out 6.0 grams of the solid white Ceteareth-20 pellets        in a 50 ml Pyrex container.    -   2. Heat the Ceteareth-20 pellets to a temperature slightly in        excess of about 100° C. (The melting point of the pellets is        about 40° C.)    -   3. Weigh out 0.06 gms of the flavonoid.    -   4. The flavonoid is added then added to the molten Ceteareth-20.    -   5. The mixture is slowly heat to temperatures in excess of        200° C. to completely solubilize the flavonoid/Ceteareth-20        mixture.    -   6. The solubilized mixture is cooled to <˜80° C. and while still        in the liquid state both water and alcohol solvents are added        and solubility observations are noted. Also, supersaturation        observations were noted by the addition of a crystal to note if        precipitation resulted.

Table XIV summarizes the experimental observations of the thermallytreated enhanced solubility observations. The ease of solution appearsto be in the following order:

-   -   Quercetin>Luteolin>Apigenin

The 3 tested flavonoids required temperatures>200° C. to fullysolubilize the 1 wt/wt % in Ceteareth-20. All flavonoid concentratedsolutions were completely soluble in ethyl alcohol. Also, all flavonoidssolutions were fully solubilized when each of the flavonoid/Ceteareth-20solutions were mixed with an equivalent volume of water. Significantincreased aqueous solubility concentrations of 3 relatively aqueousinsoluble flavonoids in Cetearth-20 resulted via the thermal treatmentprocess.

TABLE XIV A Summary of the Flavonoid/Ceteareth-20 Solubility ResultsConc. In MP Ceteareth-20 FLAVONOID MW (° C.) (mg/ml) COMMENTS APIGENIN270 ~360 ~10 The least soluble of the tested Flavonoids. Required thehighest temperature, >200° C., to solubilize the 1 wt/wt %. Not SuperSaturated at Room Temp. via crystal addition. LUTEOLIN 286 ~330 ~10 Asmall quantity dissolved at 100° C. A temperature of >200° C. requiredfor compete solubilization. Not Super Saturated at Room Temp. viacrystal addition QUERCETIN 302 ~315 ~10 Temperatures >200° C. wererequired for complete solubilization. Not Super Saturated at Room Temp.via crystal addition.

Example 7. Aqueous Solubility Comparisons of Aqueous Insoluble PlanarCyclic Organic Compounds in Polysorbate 80—with and without theFormation of Concentrates Via the Thermal Treatment Process

Mefenamic Acid, Luteolin and Apigenin, aqueous insoluble planar aromaticcompounds, were solubilized in Polysorbate 80 at elevated temperaturesto form stable soluble concentrates that when cooled to ambienttemperatures resulted in soluble concentrations that were not supersaturated and far exceeded anticipated saturation concentrations.Testing for supersaturation was evaluated by the addition a crystal tothe solution at room temperature. Water was added to these concentratesand their aqueous solution characteristics noted. For comparativepurposes, the same quantities of ingredients in the concentrates wereseparately added to the same quantity of water prior to heating to nearboiling. After cooling to room temperatures, visual observations of boththe Polysorbate 80 concentrate and non-concentrate methods werecompared.

When similar quantities of mefenamic acid, luteolin and apigenin andpolysorbate 80 that were used to prepare the concentrate were added to17 ml of water and heated to near boiling temperatures, opaque solutionswere formed. When the solutions were cooled to ambient temperatures,most of the mefenamic acid, luteolin and apigenin did precipitate fromthe aqueous solutions.

The data on the order and manner in the mixing of polysorbate 80 withrelatively aqueous insoluble cyclic organic compounds to enhance theirsolubility in aqueous solutions indicates that the compounds initiallybe heated and solubilized in polysorbate 80 to form a concentrate priorto being dissolved in water.

TABLE XV Polysorbate 80 Test Results| Prepared Thermal LiteratureTreatment Solubility in Concentration in Soln. Comments when 17 ml MP-H₂O @ 25° C. Polysorbate 80 Temp. of H₂O was added to 3 CHEMICALClassification MW ° C. (mg/ml) (mg/ml) (° C.) gms of the concentrateMEFENAMIC Active Agent 241 230 0.004 10 ~140 A clear soln formed. ACIDNot Super Sat. when cooled to Room Temp. LUTEOLIN Flavonoid 286 330 0.1450 ~250 A clear soln formed. (A Flavone) Not Super Sat. when cooled toRoom Temp. APIGENIN Flavonoid 270 360 0.001 30 ~270 A clear soln formed.(A Flavone) Not Super Sat. when cooled to Room Temp.

Example 8. Aqueous Solubility Comparisons of Relatively AqueousInsoluble Planar Cyclic Organic Compounds in Nonoxynol-9—with & withoutthe Formation of Nonoxynol-9 Concentrates Via the Thermal TreatmentProcess

Solubility testing with nonoxynol-9, a commonly utilized nonionicsurfactant in cosmetic and cleaning products, was studied as asolubilizing agent for the relatively aqueous insoluble planar aromaticcompounds such as mefenamic acid (an active agent), luteolin (aflavonoid) in addition to apigenin. The mefenamic acid, luteolin andapigenin were solubilized in nonoxynol-9 at elevated temperatures toform stable soluble concentrates that when cooled to ambienttemperatures resulted in soluble concentrations that were notsupersaturated and exceeded anticipated saturation concentrations. Theconcentrates were added to water and their aqueous solutioncharacteristics noted. For comparative purposes, the same quantities ofingredients in the concentrates were separately added to the same volumeof water prior to heating to near boiling. After cooling to roomtemperatures, visual observations of both the concentrates andnon-concentrate methods were compared.

Table XVI summarizes the results of the prepared nonoxynol-9concentrations, the temperatures required for solubilization andobservations relating to the clarity of the aqueous concentratesolutions.

TABLE XVI Nonoxynol-9 Test Results Literature Prepared ThermalSolubility in Treatment H₂O @ Concentration in Soln. Comments when 17 mlMP 25° C. Nonoxynol-9 Temp. of H₂O was added to 3 CHEMICALClassification MW (° C.) (mg/ml) (mg/ml) (° C.) gms of the concentrateMEFENAMIC Active Agent 241 230 0.004 10 ~140 A clear soln formed. ACIDNot Super Sat. when cooled to Room Temp. LUTEOLIN Flavonoid 286 330 0.1450 ~250 A clear soln formed. (A Flavone) Not Super Sat. when cooled toRoom Temp. APIGENIN Flavonoid 270 360 0.001 30 ~270 A clear soln formed.(A Flavone) Not Super Sat. when cooled to Room Temp.

When similar quantities of mefenamic acid, luteolin and apigenin andnonoxynol-9 that were used to prepare the concentrate were added to 17ml of water and heated to near boiling temperatures, opaque solutionswere formed. When the solutions were cooled to ambient temperatures,most of the mefenamic acid, luteolin and apigenin did precipitate fromthe aqueous solutions.

The data on the order and manner in the mixing of nonoxynol-9 withrelatively aqueous insoluble cyclic organic compounds to enhance theirsolubility in aqueous solutions indicates that the compounds initiallybe heated and solubilized in nonoxynol-9 to form a concentrate prior tobeing dissolved in water.

Example 9. Cholesterol Solubility Tests

The rings of cholesterol are composed of saturated hydrocarbon ringsbecause each corner of the ring is composed of a carbon atom, with twohydrogen atoms extending off the ring. The flavonoids are primarilycomposed of cyclic planar unsaturated aromatic rings while cholesterolcontains cyclic planar saturated rings. Significantly, most steroidsshare the cholesterol ring structure.

Aqueous solubility testing of Cholesterol with the nonionic surfactantsPS80 and Nonoxynol-9 and the solvent PEG 400 via the formation ofconcentrates by the thermal treatment process and the non-concentratemethod were conducted.

The materials used for this testing included:

-   -   Super-refined grades of PS80 and PEG 400 were obtained from        Croda Inc of Edison, N.J.    -   Nonoxynol-9 was obtained from Spectrum Chemical, New Brunswick,        N.J.    -   Cholesterol was obtained from Sigma-Aldrich, St. Louis, Mo.,        —with a purity of >99%.

Cholesterol properties and the concentrations of the preparedconcentrates are summarized in Table XVII.

TABLE XVII Cholesterol Test Results Prepared Literature Prepared ThermalThermal Solubility Prepared Thermal Treatment Treatment in H₂O @Treatment Concentrate in Concentrate in MP 25° C. Concentrate inNonoxynol-9 PEG-400 CHEMICAL MW ° C. (mg/ml) PS80 (mg/ml) (mg/ml)(mg/ml) Cholesterol 387 150 0.0001 10 10 10 NOTE: Cholesterol is listedas soluble in benzene, chloroform, ether, hexane, isopropyl myristate,acetone & methanol

Cholesterol solubility testing in PS80, nonoxynol-9 and PEG 400 byinitially forming concentrates of the solvents included the followingsteps:

-   -   30 mg of Cholesterol was added to 2.97 grams of PS80,        nonoxynol-9 and PEG 400 which were contained in separate 50 ml        beakers    -   The mixtures were heated about 120° C. and 80° C. for the PS80,        nonoxynol-9 and PEG 400 compositions, respectively.    -   Testing for supersaturation at room temperature was evaluated by        the addition of a crystal to the solution to determine if        precipitation occurred.    -   Visual observations of the clarity of the Cholesterol aqueous        solutions in PS80, nonoxynol-9 and PEG 400 after the addition of        17 ml of water to each of the concentrates were made more than        50 hours after cooling to room temperature.

The PS80 and nonoxynol-9 aqueous solutions of Cholesterol remained clearmore than 50 hours after cooling the solutions to ambient temperatures.However, when the 17 ml of water was added to the Cholesterol/PEG 400concentrate, a cloudy opaque solution resulted. It should be noted thatafter the addition of a few mls of water a clear solution resulted.

For comparative purposes, similar quantities of ingredients present inthe Cholesterol concentrates were separately added to the same volume ofwater prior to heating to near boiling.

Aqueous solution testing included the following steps:

-   -   In separate 50 ml beakers, 30 mg of Cholesterol, and 17 ml of        water were added to 2.97 grams of PS80, Nonoxynol-9 and PEG 400        which were contained in separate 50 ml beakers.    -   Each solution was heated to about 100° C. while mixing.    -   Visual observations of the clarity of each of the Cholesterol        containing solutions were made after the solutions were cooled        to room temperatures.

The PS80, Nonoxynol-9 and PEG 400 Cholesterol solutions resulted inopaque cloudy solutions.

This Example demonstrates that the compounds should be heated andsolubilized in surfactants such as PS80 and nonoxynol-9 to form aconcentrate, prior to the addition of water.

Example 10—Solubility Studies of Apigenin in Propylene Glycol andEthylene Glycol

Apigenin is known to be only sparsely soluble in hydrophilic andlipophilic surfactants tested (0.15-0.68 mg/ml) at 25° C. (Li et al.,Journal of Pharmaceutical Sciences, Vol. 86, No. 6, June 1997). Sincethe thermal treatment methods of surfactants herein disclosed resultedin significantly enhanced aqueous and lipophilic phase solubilityenhancements, it was decided to perform experiments to determine thesolubility properties/characteristics of apigenin in polypropyleneglycol (PG) and ethylene glycol (EG) which are surfactant solvents usedas precursors to manufacture some of the surfactants evaluated above.Observations resulting from the exposure of apigenin solubilized in PGand EG were made.

Propylene Glycol is a colorless, nearly odorless, clear, viscous liquidwith a faintly sweet taste, hygroscopic and miscible with water. PG is asolvent in many pharmaceuticals, including oral, injectable andformulations. It as is used as a humectant for many applications and asa moisturizer in medicines, cosmetics, food, toothpaste, shampoo,mouthwash hair care and tobacco products. Its boiling point is 188° C.and its molecular weight is 76 gm/mole. Propylene glycol is the basesubunit comprising the polypropylene glycol surfactants (PPGs).

Propylene Glycol's structural formula is:

Ethylene glycol, is a colorless, oily liquid possessing a sweet tasteand mild odor. Its molecular weight is 62 and its boiling point 197° C.Ethylene glycol is the base subunit comprising the polyethylene glycolsurfactants (PEGs).

Ethylene glycol's structural formula is:

The solubility testing for both PG & EG were performed as follows:

-   -   In separate 18 ml vials, 200 mg of Apigenin was added to 4.80        gram of PG and also to EG. Both vials were heated to about        175° C. for the PG vial & to about 190° C. for the EG vial.        Apigenin was completely solubilized in both vials to form 40        mg/ml solutions.    -   Upon cooling to room temperature, appreciable apigenin        precipitation was observed for both PG and EG solutions. Thus        demonstrating that these solutions were supersaturated.    -   The apigenin/PG and the apigenin/EG mixtures were then        centrifuged for 20 minutes at 3,600 rpm; resulting in a pale        yellow clear liquid and substantial fraction of precipitated        apigenin that had separated out of both solutions.

In addition, after a few drops of water were added to the saturatedsolubilized apigenin/PG and EG solutions, a significant fraction of thesolubilized apigenin precipitated out of both solutions thus limitingtheir usefulness for subsequent aqueous formulation development.

Example 11—Solubility of Flavonoid Surfactant and PEG 400 Concentratesin Oil Solvents

The primary objective of this study was to evaluate enhancement ofplanar ring structured organic compounds solubility concentrations inoil. In general, nonionic surfactants are characterized by hydrophilichead groups that do not ionize appreciably in water. Examples includepolyoxyethylenated alkylphenols, alcohol ethoxylates, alkylphenolethoxylates, and alkanolamides. Nonionic surfactants tend to be goodsolubilizers and are relatively nontoxic. They are usually easilyblended with other types of surfactants (i.e., used as cosurfactants)and therefore have found widespread use in cosmetic, pharmaceutical andenvironmental applications. The performance of nonionic surfactants,unlike anionic surfactants, is relatively insensitive to the presence ofsalts in solution.

The Hydrophile-Lipophile Balance (HLB) number is an indication of therelative strength of the hydrophilic and hydrophobic portions of themolecule and can be used to characterize the relative affinity ofsurfactants for aqueous and organic phases. A high HLB number generallyindicates good surfactant solubility in water, while a low HLB numberindicates a lower aqueous solubility and higher relative affinity forthe organic phase. A surfactant with a low HLB number can partitionsignificantly into the organic phase and form reverse micelles havinghydrophilic interiors and lipophilic exteriors.

Polyglyceryl-4 Oleate (CAS #68605-19-6), nonionic surfactant with a HLBvalue of 5 suggests suitability for enhancing the solubility of planarring structured organic compounds for oil based topical formulationswhile Polysorbate 80 as noted in Example 4 with an HLB value of 15should be most appropriate for solubilizing apigenin in oil-in-watertopical formulations.

Polyglycerol-4 Oleate obtained from Making Cosmetics (Renton, Wash.) isa distilled triyglycerol ester based on vegetable oleic acid and isPEG-free. It disperses in water and is well suited for water-in-oilemulsions). Borage oil was obtained from Sigma-Aldrich (St. Louis, Mo.)and Mineral Oil, USP, Canola Oil and Jojoba Oil from Spectrum Chemicalof New Brunswick, N.J.

Tables XVIII, XIX & XX summarize visual observation when concentrates ofluteolin in polyglcyeryl-4 oleate and apigenin in polysorbate 80 andPEG-400 were added to a variety of oils. It should be noted thatluteolin and apigenin are insoluble in the test oils and that the 10mg/ml luteolin concentrate in polyglcyeryl-4 oleate was notsupersaturated at room temperature as determine by the absence of aprecipitate upon the addition of a luteolin crystal to theluteolin/polyglcyeryl-4 oleate concentrate.

The lower HLB valued emulsifiers are better in water in oil as they aremore lipophilic; the higher valued HLB emulsifiers are more hydrophilic

TABLE XVIII Solubility Observations of a Luteolin/Polyglyceryl-4 OleateConcentrate in Oils Observations when 1 ml of a 10 mg/ml Luteolin/Polyglyceryl-4 Oleate Concentrate was added to OIL 5 ml of DifferentOils Mineral Oil Totally Miscible Jojoba Totally Miscible Canola TotallyMiscible Borage Totally Miscible

TABLE XIX Solubility Observations of an Apigenin/Polysorbate 80Concentrate in Oils Observations when 1 ml of a 30 mg/mlApigenin/Polysorbate OIL 80 Concentrate was added to 5 ml of DifferentOils Mineral The PS80 phase containing the solubilized Apigeninseparated Oil out from the Mineral oil phase - minimal solubilization ofthe PS80 phase in the Oil phase noted Jojoba Similar to the Mineral OilObservations Canola Similar to the Mineral Oil Observations BorageSimilar to the Mineral Oil Observations

TABLE XX Solubility Observations of an Apigenin/PEG-400 Concentrate inOils Observations when 1 ml of a 30 mg/ml Apigenin/PEG-400 OILConcentrate was added to 5 ml of Different Oils Mineral Oil Two distinctclear phases resulted with the PEG-400 containing solubilized Apigeninsettle out Jojoba Similar to the Mineral Oil Observations Canola Similarto the Mineral Oil Observations Borage Similar to the Mineral OilObservations.

Example 12. Capsaicin Solubility Tests

Aqueous solubility testing of capsaicin (a pain reliever) with thenonionic surfactants PS80 and the solvent PEG 400 via the formation ofconcentrates by the thermal treatment process and the non-concentratemethod were compared.

The materials used for this testing included:

-   -   Super-refined grades of PS80 and PEG 400 were obtained from        Croda Inc of Edison, N.J.    -   Capsaicin was obtained from Sigma-Aldrich, St. Louis, Mo.,        Product #360376.

Capsaicin properties and the concentrations of the prepared concentratesare summarized in Table XXI.

TABLE XXI Properties of Capsaicin Prepared Prepared Literature ThermalThermal Solubility Treatment Treatment in H₂O Concentrates ConcentratesMP @ ° C. in PS80 in PEG-400 CHEMICAL MW (° C.) (mg/ml) (mg/ml) (mg/ml)Capsaicin 305 65 <0.01 40 40 NOTE: Merck lists Capsaicin as insolublewater but soluble in benzene, chloroform, ether, and alcohol.

Capsaicin solubility testing in PS80 and PEG 400 by initially formingconcentrates using the thermal treatment method:

-   -   30 mg of capsaicin was added to 2.97 grams of PS80 and PEG 400        which were contained in separate 50 ml beakers    -   These mixtures were heated to about 50° C. while mixing.    -   Addition of a few crystals of capsaicin to the cooled liquids        did not result in precipitation; thereby demonstrating that        these liquid is not supersaturated.    -   Visual observations of the clarity of the capsaicin aqueous        solutions in PS80 and PEG 400 upon the addition of 17 ml of        water to each of the capsaicin concentrates were made after more        than 50 hours subsequent to cooling to room temperature.

A clear solution of the PS80 concentrate resulted after the addition of17 ml of H₂O and remained so more than 50 hours after the solutioncooled to room temperature. However, a clear solution resulted afteronly the addition of a few mls of water (about 2 to 4 mls) to thecapsaicin/PEG 400 concentrate which became opaque and cloudy with theaddition of 17 ml of H₂O.

For comparative purposes, similar quantities of ingredients present inthe Capsaicin concentrates were separately added to the same volume ofwater prior to heating to near boiling.

Aqueous solution testing included the following steps:

-   -   In separate 50 ml beakers, 30 mg of capsaicin and 17 ml of water        were added to 2.97 grams of PS80 and PEG 400 which were        contained in separate 50 ml beakers.    -   Each solution was heated to about 100° C. while mixing.    -   Visual observations of the clarity of the capsaicin aqueous        solutions in PS80 and PEG 400 subsequent to the addition of 17        ml of water to each of the capsaicin concentrates were made more        than 50 hours after cooling to room temperature.

The PS80 and PEG 400 capsaicin solutions resulted in opaque cloudysolutions.

This Example demonstrates that these compounds be heated and solubilizedin surfactants such as PS80 to form a concentrate, prior to the additionof water.

Example 13. Coenzyme Q10, Polysorbate 80 and Jojoba Oil SolubilityTesting

Since it was experimentally observed that the thermal process of formingconcentrates (made from nonionic surfactants such as PS80 and a varietyof organic compounds containing planar aromatic ring structures), iseffective in enhancing aqueous solubility, solubility testing withCoQ10, a planar aromatic ring structured compound with good oilsolubility, together with PS80 and an oil such as Jojoba, was conductedto provide additional data.

Coenzyme Q10, an oil-soluble vitamin-like substance, is present in mostcells primarily in the mitochondria. It is a component of the electrontransport chain and participates in aerobic cellular respiration,generating energy in the form of ATP. Ninety-five percent of the humanbody's energy is generated this way.

Jojoba oil is a mixture of wax esters, 36 to 46 carbon atoms in length.Each molecule consists of a fatty acid and a fatty alcohol joined by anester bond. 98% of the fatty acid molecules are unsaturated at the 9thcarbon-carbon bond.

Jojoba oil, Coenzyme Q10 and polysorbate 80 were obtained were obtainedfrom Making Cosmetics, Renton, Wash.

Initial testing (Test 1) verified the immiscibility of the Jojoba andwater oil phases. Accordingly,

-   -   6 ml of Jojoba oil and 20 ml of water were thoroughly mixed in a        30 ml beaker.    -   After more than 2 hours, it was observed that the pale yellow        colored Jojoba oil and water phases separated. The water phase        was clear and also colorless indicating that the pale yellow        jojoba oil was insoluble in the aqueous phase.

The next test (Test 2) was conducted as follows:

-   -   0.3 grams of the orange CoQ10 powder was added to 1 gram of PS80        and heated to about 50° C. The CoQ10 dissolved in the PS80        forming a red/orange colored solution. The CoQ10 remained        solubilized and remained solubilized when the mixture was cooled        to room temperature.    -   Testing for supersaturation at room temperature was evaluated by        the addition of a crystal to the solution to determine if        precipitation occurred. It was observed that the CoQ10/PS80        concentrate was not supersaturated.    -   6 ml of Jojoba oil was mixed with 20 ml of water and the mixture        was thoroughly stirred while heated to about 50° C.    -   The water in oil mixture at 50° C. was then added to the        CoQ10/PS80 concentrate and thoroughly stirred for about 1        minute.    -   After a little more than 2 hours, it was noted that the water        phase contained the CoQ10 as observed by the clear red/orange        colored water phase. The Jojoba oil phase separated from the        aqueous phase and appeared to have little, if any, dissolved        CoQ10 as observed by the pale yellow colored jojoba oil phase        distinct from the red/orange color of the aqueous phase        indicative of the solubilized CoQ10/PS80 concentrate.

The final testing (Test 3) with CoQ10, Jojoba oil and water was carriedout as follows:

-   -   0.3 grams of the CoQ10 powder were dissolved in 6 ml of Jojoba        oil at room temperature. To this mixture was added 1 gram of        PS80 and the resulting mixture was then heated to about 50° C.    -   20 ml of water which was preheated to about 50° C. was added to        this mixture. The combined mixture was thoroughly stirred for 1        minute.    -   After about 2 hours, it was visually observed that most of the        CoQ10 did separate from the aqueous phase and was mostly        concentrated in the red/orange Jojoba oil phase. Also, the    -   aqueous phase was partially opaque and quite unlike the clear        aqueous phase of the beaker in Test 2 which contained the bulk        of the red/orange solubilized CoQ10.

As a consequence of these tests, it is concluded that the poorly solubleCoQ10 initially be solubilized in PS80 to form a concentrate in order toenhance its solubility in aqueous solutions.

Example 14—Preparation of Topical Formulations of Apigenin UsingApigenin/Surfactant Concentrates

Active agent concentrates can be used to formulate compositions with ahigher concentration of dissolved active agent in the preparedformulation than that achievable without the use of making the activeagent concentrates first. Multiple prototype solution, gel, ointment,and emulsion topical formulations containing Apigenin in the dissolvedstate were prepared using a Apigenin/Polysorbate-80 concentrate and anApigenin/PEG300 concentrate. These formulations are summarized in TablesXXII & XXIII.

TABLE XXII Prototype Solution, Gel and Ointment, Formulations Preparedwith Apigenin/Surfactant Concentrates Apigenin dissolved in PEG300Apigenin Apigenin dissolved in PS80 Concentrate Concentrate dissolved inPS80 Anhy- Anhydrous Anhy- Anhy- Concentrate drous (EtOH + Aqueous drousdrous Aqueous (DMSO/ Oily (NO PEG PEG gel Control EtOH) enhancers) EtOH)Aqueous Solution Ointment (modified) solution Component % w/w Apigenin(API) Apigenin/PS80 concentrate 9 9 9 9 9 9 9 9 9 (5.6% w/w APIGENIN)Apigenin/PEG300 14.3 14.3 concentrate (3.5% w/w APIGENIN) Dimethylsulfoxide 45 45 (DMSO) Polysorbate 80 (PS80) 21 10 Polyethylene glycol300 14.7 (PEG 300, IIG max-57%) PEG 200 (IIG max-39%) 39 13.7 PEG 3350(IIG max 40%) 40 Span 80 7 Water 34 46 33 36 72 51.5 54 150 mM NaClEthanol (EtOH) 46 66 45 Propylene glycol (PG) 10 20 20 20 Isopropylmyristrate (IPM) Transcutol (TC) 25 25 25 25 Dimethyl isosorbide (DMI)Hexylene glycol (HG) 12 12 12 12 12 12 12 Myristyl lactate (ML) 10 Oleicacid (Oac) 5 Oleyl alcohol (OA) Sodium hyaluronate 0.5 (NaH, Ultra lowmolecular weight) Sodium hyaluronate 2.0 (NaH, High molecular weight[e.g. >100 KDa]) TOTAL 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0100.0 100.0 100.0

TABLE XXIII Emulsion Formulations Prepared with Apigenin/SurfactantConcentrates Emulsions (0.25% w/w Apigenin) Emulsifying Pemulen Wax BaseBase Formulation # 17 18 Component % w/w Apigenin 5.6%/PS80 Concentrate4.5 4.5 Transcutol, USP 10 10 Methylparaben, NF 0.17 0.17 Propylparaben,NF 0.03 0.03 EDTA, USP 0.1 0.1 Purified water QSad Carbopol 980 0.4 0.4Emulsifying wax, NF 12 Pemulen TR1 0.3 White petrolatum, NF 5 Myristyllactate, NF 5 12.2 Cyclomethicone, NF 2 10 Oleyl alcohol, NF 2 2Cholesterol, NF 1 Butylated hydroxytoluene, NF (BHT) 0.1 0.1 Dilute HClsolution, NF QSad pH7 QSad pH7 10% NaOH Solution Total 100 100

Example 15—In Vitro Percutaneous Absorption of Apigenin fromFormulations Using Human Skin

Overall, data from this in vitro skin permeation experiment indicatedthat DPSI prototypes exhibited a range of delivery profiles from variousformulation bases with contrasting organoleptic/cosmetic properties.

The purpose of this study was to characterize in vitro percutaneousabsorption of Apigenin from topical formulations following applicationto excised human skin from elective surgery.

This study was conducted using procedures adapted from the FDA and AAPSReport of the Workshop on Principles and Practices of In VitroPercutaneous Penetration Studies: Relevance to Bioavailability andBioequivalence (Skelly et al., 1987). Human tissue from a single donorwas dosed with 5 mg/cm² of formulation. The compositions of all theformulations evaluated in this study are summarized in Table XXIV andXXV.

The clinically relevant dose of 5 mg/cm² was applied to dermatomed humanabdominal tissue from a single donor obtained following electivesurgery. The thickness of the tissue ranged from 0.034 inches (0.870 mm)with a mean+/−standard deviation in thickness of +/−0.005 inches(+/−0.131 mm) and a coefficient of variation of 15%.

Percutaneous absorption was evaluated using this human abdominal tissuefrom a single donor mounted in Bronaugh flow-through diffusion cells.The cells were maintained at a constant temperature of 32° C. by use ofrecirculating water baths. These cells have a nominal diffusion area of0.64 cm². Fresh receptor phase (PBS, pH 7.4, containing 0.1% sodiumazide and 4% Bovine Serum Albumin) was continuously pumped under thetissue at a flow rate of nominally 0.25 ml/hr and collected in 6-hourintervals. The receptor phase samples were collected in pre-weighedscintillation vials; the post weights were taken at the end of thestudy. Following the 24-hour duration exposure, the formulation residingon the tissue surface was removed by tape-stripping with CuDerm D-Squamestripping discs. The epidermis, dermis, and receptor phase samples werelabeled and frozen prior to subsequent analysis of Apigenin content byLC-MS/MS and ultimate sample disposal.

Generally, permeation of Apigenin into the receptor compartment was low.However, relatively high permeation into the receptor phase was achievedwhen Apigenin was applied in neat DMSO. Moderate dermal deposition wasobserved while relatively high epidermal values were associated with themajority of the compositions evaluated. Overall, DMSO based solutions,3530-14A, -15B and -31 (neat DMSO control) produced the highest tissuepenetration and deposition. An anhydrous solution (3530-14B,EtOH/PG/IPM/ML/Oleic acid) also offered relatively favorable deliverycharacteristics. Although formulation 3530-14B generated lower tissuepenetration and deposition to than the DMSO based solutions it may offera viable alternative. As anticipated, the PEG based solution producedgreater delivery than the PEG ointment.

Several semi-solid prototypes were evaluated and included an aqueous gel(3530-22), an aqueous gel based nano-suspension (3530-24), two emulsions(3530-29 [Emulsifying wax base] and -30 [Pemulen base]) and a PEGointment (3530-18B). It was notable that the presence of Sodiumhyaluronate polymer in the aqueous gel did not significantly impairreceptor penetration and dermal deposition when compared to theanalogous solution control. In terms of delivery efficiency (% ofapplied dose) of Apigenin, the Pemulen emulsion offered slightly morefavorable delivery characteristics relative to a solubilizedApigenin/Sodium hyaluronate based gel (3530-22). When amount deliveredwas considered, the aqueous gel (3530-22) generated higher tissuedeposition than the Pemulen emulsion. However, the Pemulen emulsiongenerated the highest receptor levels of the semi-solid compositionstested, when efficiency and amount delivered were evaluated.

FIG. 3 is a graphical illustration of the epidermal, dermal, receptorfluid and total penetration apigenin profiles as a percentage of theapplied apigenin dosage content for the several topical formulations.

TABLE XXIV SOLUTION AND GEL COMPOSITIONS Apigenin dissolved in Apigenindissolved in Nano- Apigenin dissolver in PS80 Concentrate PEG300Concentrate PS80 Concentrate suspension Anhydrous Anhydrous AnhydrousAnhydrous Aqueous Aqueous Apigenesis (DMSO/ (EtOH + Oily PEG PEG gelControl gel DMSO Formulation concepts EtOH) enhancers) Aqueous SolutionOintment (modified) solution (modified) Control Formulation # 1 2″ 4 910 13 14 15 n/a Formulation ID: 3530- 14A 14B 15B 18A 18B 22 23 24 31Component % w/w Apigenin (API) 0.5 1.5 Apigenin/PS80 concentrate 9 9 9 99 (5.6% w/w Apigenin) Apigenin/PEG300 concentrate 14.3 14.3 (3.5% w/wApigenin) Dimethyl sulfoxide (DMSO) 45 45 98.5 Polysorbate 80 (PS80)Polyethylene glycol 300 14.7 (PEG 300, ||G max-57%) PEG 200(||G max-39%)39 13.7 PEG 3350 (||G max 40%) 40 Span 80 Water 46 51.5 54 60 150 mMNaCl Ethanol (EtOH) 46 66 Propylene glycol (PG) 10 20 20 Isopropylmyristate (IPM) Transcutol (TC) 25 25 25 Dimethyl isosorbide (DMI)Hexylene glycol (HG) 12 12 12 12 12 Myristyl lactate (ML) 10 Oleic acid(OAc) 5 Oieyl alcohol Sodium hyaluronate (NaH, Ultra 0.5 0.5 lowmolecular weight) Sodium hyaluronate (NaH, High 2.0 2.0 molecular weight[e.g. >100 KDa]) TOTAL 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0100.0 Formulation Labels A B C D E F G H K

TABLE XXV EMULSION COMPOSITIONS Emulsions (0.25% w/w Apigenin)Emulsifying Pemulen Apigenesis Formulation concepts Wax Base BaseFormulation # 17 18 Formulation ID: 3530- 29 30 Component % w/w Apigenin5.6%/PS80 Concentrate 4.5 4.5 Transcutol P 10 10 Methylparaben, NF 0.170.17 Propylparaben, NF 0.03 0.03 EDTA, USP 0.1 0.1 Purified water QSadCarbopol 980 0.4 0.4 Emulsifying wax, NF 12 Pemulen TR1, NF 0.3 Whitepetrolatum, NF 5 Myristyl lactate 5 12.2 Cyclomethicone 2 10 Oleylalcohol 2 2 Cholesterol, NF 1 Butylated hydroxytoluene, NF (BHT) 0.1 0.1Dilute HCl solution, NF QSad pH 6.5 10% NaOH Solution Total 100.0 100.0Formulation Labels

I J

Example 16—In Vitro Irritancy Determination of Apigenin Formulations

The purpose of this study was to compare the acute dermal irritationpotential of 5 Apigenin containing formulations using the Epiderm MTTET₅₀ assay. The time required for atest article to reduce tissueviability to 50%, i.e. ET₅₀, correlates well with the traditional invivo rabbit skin irritation test

This study was conducted at MB Research Laboratories (Spinnerstown, Pa.to conduct the EpiDerm MTT Assay. EpiDerm tissue was incubated with ApiGenesis formulations for 1, 4 and 24 hours (two tissues per time pointfor each test article). Triton X-100, 1%, was used as positive control.After incubation, the tissues were rinsed to remove test articles andcytotoxicity was determined with MTT dye[3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolylblue]. Only viable cells are capable of enzymatic reduction of MTT intoa purple formazan product that is quantitatively measured followingextraction from the tissue. Dead cells do not reduce MTT and thus, thisassay allows determination of the effect of test article exposure ontissue viability when compared to untreated control tissues.

Tissue viability data at the three incubation time points were used tocalculate the time required to reduce tissue viability to 50% (EffectiveTime, ET₅₀) for each formulation. The ET₅₀ is well correlated with thetraditional Draize rabbit skin irritation test dermal scores and used topredict irritancy class (Table XXVI).

TABLE XXVI Epiderm MTT ET₃₀ assay-in vivo Draize Irritancy CorrelationET₅₀ (h) Expected in vivo Irritancy <0.5 Severe, Probable corrosive0.5-4  Moderate  4-12 Moderate to Mild 12-24 Very Mild >24Non-Irritating

Table XXVII presents the ranking of test articles based on unauditedET₅₀ values and their predicted in vivo irritancy classification. Theseformulations ranged from non-irritants to mild-to-moderate irritants.

TABLE XXVII Formulations Irritancy Classification Formulation ID (referto “Formulation Predicted in Labels” on Tables XXIV vivo Irritancy andTable XXV) ET₅₀ (h) Classification (J) 3530-30 >24.0 Non-Irritating (C)3530-15B >24.0 Non-Irritating (F) 3530-22 12.6 Very Mild (H) 3530-2411.11 Mild to Moderate (B) 3530-14B 8.0 Mild to Moderate

The formulations can be ranked, based on ET50 values, as follows:[non-irritant] (J)=(C)<F≈(H)<(B) [mild-to-moderate]

The ET50 for the positive control 1% Triton X-100, was 5.9 hours and waswithin the manufacturer's historical range for the assay, whichconfirmed that the test system is sensitive to irritants.

In summary, the In Vivo irritancy Determinations of the 5 formulationsranged from non-irritating to mild-to-moderate irritating and areconsidered to have low acute skin irritation potential.

It should be understood that a wide range of changes and modificationscould be made to the embodiments described above. It is thereforeintended that the foregoing description illustrates rather than limitsthis invention, and that it is the following claims, including allequivalents, which define this invention.

All documents and references cited above are hereby incorporated byreference in their entirety in this application.

While the invention has been described with reference to an exemplaryembodiment, it will be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted forelements thereof without departing from the scope of the invention. Inaddition, many modifications may be made to adapt a particular situationor material to the teachings of the invention without departing from theessential scope thereof. Therefore, it is intended that the inventionnot be limited to the particular embodiment disclosed as the best modecontemplated for carrying out this invention, but that the inventionwill include all.

The invention claimed is:
 1. A composition comprising: a concentrateformed by mixing a polyphenol with a non-ionic surfactant, in theabsence of a carrier, to form a mixture, and heating said mixture to atemperature of greater than 150° C. to form said concentrate, whereinupon cooling to room temperature the concentrate is not supersaturated,and the concentration of said polyphenol is greater than the saturationconcentration of said polyphenol in said non-ionic surfactant.
 2. Thecomposition as in claim 1, wherein said non-ionic surfactant is apolysorbate.
 3. The composition as in claim 1 further comprisinghyaluronic acid.
 4. The composition as in claim 1, wherein saidpolyphenol and said non-ionic surfactant are heated to a temperature ofgreater than 170° C.
 5. The composition as in claim 1 further comprisinga carrier.
 6. The composition as in claim 5, wherein said carrier is awater based carrier.
 7. The composition as in claim 5, wherein saidcarrier comprises dimethyl sulfoxide and water.
 8. The composition as inclaim 5, wherein said carrier comprises dimethyl sulfoxide,hydroxypropyl cellulose and water.
 9. The composition as in claim 5,wherein said carrier comprises ethanol, propylene glycol and water. 10.The composition as in claim 5, wherein said carrier comprises ethanol,propylene glycol, water and a gelling agent selected from the groupconsisting of hydroxyethyl cellulose, sodium hyaluronate and acrosslinked polyacrylate polymer.
 11. The composition as in claim 5,wherein said carrier comprises an oil phase, a surfactant, and water.12. The composition as in claim 11, wherein said oil phase comprisesethoxydiglycol, myristyl lactate, cyclomethicone or oleyl alcohol. 13.The composition as in claim 5, wherein said carrier is an oil basedcarrier.
 14. The composition as in claim 5, wherein said carriercomprises an alcohol, ethoxydiglycol, propylene glycol, hexylene glycol,butylene glycol, dipropylene glycol, glycerin, water, saline, DMSO,isopropyl myristate, mineral oil, a surfactant, or dimethyl isosorbide.15. The composition as in claim 1, wherein said non ionic surfactant ispolysorbate 80, polyoxy 20 cetostearyl ether, or polyoxyl 40hydrogenated castor oil.
 16. The composition as in claim 1, wherein saidcomposition is in the form of a pharmaceutical.
 17. The composition asin claim 1, wherein said composition is in the form of a nutraceutical,cosmeceutical, food or medical food.
 18. The composition as in claim 1,wherein said composition is in the form of a beverage.
 19. Thecomposition as in claim 1, wherein said composition is in the form of asolution or emulsion.
 20. The composition as in claim 1, wherein saidcomposition is in the form of a gelatin capsule or enteric coatedcapsule.
 21. The composition as in claim 1, wherein said composition isin the form of a liquid, serum, cream, lotion, gel, spray, foam,ointment or cleanser.
 22. The composition as in claim 1, furthercomprising one or more additive selected from the group consisting ofhyaluronic acid, a preservative, a buffer, a humectant, ananti-inflammatory agent, an emollient, a moisturizer, a thickeningagent, and an analgesic.